Combination of oncolytic virus with immune checkpoint modulators

ABSTRACT

The present invention provides a combination comprising at least an oncolytic virus and one or more immune checkpoint modulator(s) for use for the treatment of a proliferative disease such as cancer. It also relates to a kit comprising an oncolytic virus and one or more immune checkpoint modulator(s) in separate containers. It also concerns a pharmaceutical composition comprising effective amount of an oncolytic virus and one or more immune checkpoint modulator(s).

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a U.S. National Stage Application pursuant to 35U.S.C. § 371 of International Patent Application PCT/EP2015/066353,filed on Jul. 16, 2015, and published as WO 2016/009017 on Jan. 21,2016, which claims priority to European Patent Application 14306155.4,filed on Jul. 16, 2014, all of which are incorporated herein byreference in their entireties for all purposes.

FIELD OF THE INVENTION

The present invention generally relates to the field of oncolyticvirotherapy and more specifically to compositions and methods to treat,prevent, or inhibit proliferative diseases, especially cancer.Embodiments include an oncolytic virus for use for the treatment ofcancer in combination with one or more immune checkpoint modulator(s).Embodiments also include a kit comprising such components and method oftreatment using said oncolytic virus with said one or more immunecheckpoint modulator(s).

Each year, cancer is diagnosed in more than 12 million subjectsworldwide. In industrialized countries, approximately one person outfive will die of cancer. Although a vast number of chemotherapeuticsexist, they are often ineffective, especially against malignant andmetastatic tumors that establish at a very early stage of the disease.Moreover, antitumor immunity is often ineffective due to the fact thattumor cells have evolved mechanisms to escape host defense. One of themajor mechanisms of immune suppression is a process known as “T-cellexhaustion”, which results from chronic exposure to antigens and ischaracterized by the upregulation of inhibitory receptors. Theseinhibitory receptors serve as immune checkpoints in order to preventuncontrolled immune reactions. Various immune checkpoints acting atdifferent levels of T cell immunity have been described in theliterature, including programmed cell death protein 1 (PD-1) and itsligands PD-L1 and PD-L2, CTLA-4 (cytotoxic T-lymphocyte associatedprotein-4), LAG3 (Lymphocyte-activation gene 3), B and T lymphocyteattenuator, T-cell immunoglobulin, mucin domain-containing protein 3(TIM-3), and V-domain immunoglobulin suppressor of T cell activation.

Whatever the mechanism of action, these immune checkpoints can inhibitthe development of an efficient anti-tumor immune response. There isincreasing interest in the possible therapeutic benefits of blockingsuch immune checkpoints as a means of inhibiting immune system toleranceto tumors and thus rescue exhausted antitumor T cells (Leach et al.,1996, Science 271: 1734-6). A vast number of antagonistic antibodieshave been developed during the last decade (e.g. anti Tim3, -PD-L1,-CTLA-4, -PD1, etc) and most importantly, some have been associated withobjective clinical responses in cancer patients. Antibodies targetingCTLA-4 are already marketed (e.g. Ipilimumab, Yervoy, Bristol-MyersSquibb, BMS) for metastatic melanoma. BMS reported that from 1800melanoma patients treated with ipilimumab 22% are still alive 3 yearslater. Antibody therapies with anti PD-L1 (e.g. MPDL3280A, Roche), antiPD-1 (e.g. Nivolumab, BMS) are also ongoing.

Another therapeutic approach that is emerging in the field of cancer isoncolytic viruses (Hermiston, 2006, Curr. Opin. Mol. Ther. 8: 322-30).Oncolytic viruses are capable of selective replication in dividing cells(e.g. cancer cell) while leaving non dividing cells (e.g. normal cells)unharmed. As the infected dividing cells are destroyed by lysis, theyrelease new infectious virus particles to infect the surroundingdividing cells. Cancer cells are ideal hosts for many viruses becausethey have the antiviral interferon pathway inactivated or have mutatedtumour suppressor genes that enable viral replication to proceedunhindered (Chernajovsky et al., 2006, British Med. J. 332: 170-2). Anumber of viruses including adenovirus, reovirus, measles, herpessimplex, Newcastle disease virus and vaccinia have now been clinicallytested as oncolytic agents.

Some viruses are naturally oncolytic (such as reovirus and the Senecavalley picornavirus) while others are engineered for tumor selectivityby modifying the viral genome. Such modifications include functionaldeletions in essential viral genes, the use of tumor- or tissue-specificpromoters to control the viral gene expression and tropism modificationto redirect virus to the cancer cell surface.

The first oncolytic virus to be approved by a regulatory agency was agenetically modified adenovirus named H101 (Shanghai Sunway Biotech)that gained approval in 2005 from China's State Food and DrugAdministration (SFDA) for the treatment of head and neck cancer. Anotheroncolytic adenovirus, named ONYX-015 is in ongoing clinical trials forthe treatment of various solid tumors (in phase III for the treatment ofrecurrent head and neck cancer) (Cohen et al., 2001, Curr. Opin.Investig. Drugs 2: 1770-5). As another example, oncolytic herpes simplex1 (T-VEC) was genetically engineered to attenuate the virus virulence,increase selectivity for cancer cells and enhance antitumor immuneresponse (through GM-CSF (Granulocyte-macrophage colony-stimulatingfactor) expression). Clinical efficacy in unresectable melanoma has beendemonstrated in Phase II and Phase III clinical trials (Senzer et al,2009, J. Clin. Oncol. 27: 5763-71).

Vaccinia viruses (VV) possess many of the key attributes necessary foruse in oncolytic virotherapy such as natural tropism for tumors, stronglytic ability, short life cycle with rapid cell-to-cell spread, highlyefficient gene expression and a large cloning capacity. In addition,they have been delivered to millions of individuals during the smallpoxeradication campaign without major safety concerns. In this respect, aTK (Thymidine Kinase) and VGF (for VV growth factor) double deleted VVexpressing GM-CSF (named JX-963) showed significant cancer selectivityin tumor bearing mice (Thorne et al., 2007, J Clin Invest. 117: 3350-8).On the same line, JX-594, a TK-deleted VV (Wyeth strain) armed withGM-CSF, has shown promising clinical data, and a randomized Phase IIItrial in hepatocellular carcinoma is expected to start soon.

Combination therapies have also been described in the literature.WO2010/014784 describes the combination of an anti CTLA4 antibody withchemotherapeutics used for treating cancer such as GLEEVEC, TAXOL andthe like. WO2014/047350 envisages a recombinant oncolytic virus with agene encoding an anti-PD-1 antibody inserted in the viral genome.

Technical Problem

One may expect that cancer will continue to be a serious global healththreat for many years due to the high number of causal factors that mayact together or separately to initiate or promote the development of acancer. Moreover, malignant and especially metastatic tumors are oftenresistant to conventional therapies explaining the significant morbidityof some cancers.

Thus, there is an important need to develop more effective approaches,for improving prevention and treatment of such proliferative diseases,and especially combination approaches.

The combination therapy, wherein an oncolytic virus and one or moreimmune checkpoint modulator(s) were both administered, provided asynergistic immune response as compared to either approach used alone.Surprisingly, the combined treatment wherein an oncolytic vaccinia viruswas administered before administration of an anti-checkpoint antibodysuch as anti-PD-1 or anti-CTLA-4, improved the anti-tumor response asevidenced in an appropriate model animal, thus potentially providing aneffective and powerful therapy against cancer. Accordingly, theembodiments provided herein provide a significant advance in thetreatment and prevention of proliferative diseases such as cancer.

This technical problem is solved by the provision of the embodiments asdefined in the claims.

Other and further aspects, features and advantages of the presentinvention will be apparent from the following description of thepresently preferred embodiments of the invention. These embodiments aregiven for the purpose of disclosure.

SUMMARY OF THE INVENTION

The present invention concerns a synergistic combination of oncolyticviruses with one or more immune checkpoint modulator(s) for use for thetreatment of proliferative diseases such as cancers. The oncolytic virusis preferably selected from the group consisting of reovirus, New CastleDisease virus (NDV), vesicular stomatitis virus (VSV), measles virus,influenza virus, Sinbis virus, adenovirus, poxvirus and herpes virus(HSV) and the like. In one embodiment, the oncolytic virus is a vacciniavirus. In a preferred embodiment, the vaccinia virus is engineered tolack thymidine kinase (TK) activity (e.g. the genome of said VV has aninactivating mutation in J2R gene or a gene deletion to produce adefective TK phenotype). Alternatively or in combination, the vacciniavirus is engineered to lack ribonucleotide reductase (RR) activity (e.g.the genome of said VV has an inactivating mutation in I4L and/or F4Lgene or a gene deletion to produce a defective RR phenotype).

In one embodiment, the vaccinia virus further expresses at least onetherapeutic gene, in particular a gene encoding a suicide gene productand/or an immunostimulatory protein.

In one embodiment, the one or more immune checkpoint modulator(s) is anantagonist molecule that antagonizes the activity of PD-1, PD-L1 orCTLA4 with a specific preference for an anti PD-1 antibody and/or ananti CTLA4 antibody.

In one embodiment, the oncolytic virus is preferably formulated forintravenous or intratumoral administration and/or the one or more immunecheckpoint modulator(s) is preferably formulated for intravenous orintraperitoneal or intratumoral administration.

The present invention further provides a method for the treatment of aproliferative disease including cancer which comprises administering toa mammal in need thereof synergistically effective amounts of anoncolytic virus as described herein and of one or more immune checkpointmodulator(s) as described herein. In one embodiment, the proliferativedisease treated by the method of the invention is cancer and especiallymelanoma, renal cancer, prostate cancer, breast cancer, colorectalcancer, lung cancer and liver cancer. In one embodiment, the methodcomprises an additional step in which a pharmaceutically acceptableamount of a prodrug is administered to said mammal. The administrationof said prodrug takes place preferably at least 3 days after theadministration of said oncolytic virus or virus composition.

The present invention further provides a kit including an oncolyticvirus as described herein and one or more immune checkpoint modulator(s)preferably in separate containers.

DETAILED DESCRIPTION

The present invention concerns a combination comprising at least anoncolytic virus and one or more immune checkpoint modulator(s) for usefor the treatment of proliferative diseases such as cancer.

Definitions

As used throughout the entire application, the terms “a” and “an” areused in the sense that they mean “at least one”, “at least a first”,“one or more” or “a plurality” of the referenced components or steps,unless the context clearly dictates otherwise. For example, the term “acell” includes a plurality of cells, including mixtures thereof.

The term “one or more” refers to either one or a number above one (e.g.2, 3, 4, 5, etc).

The term “and/or” wherever used herein includes the meaning of “and”,“or” and “all or any other combination of the elements connected by saidterm”.

The term “about” or “approximately” as used herein means within 20%,preferably within 10%, and more preferably within 5% of a given value orrange.

As used herein, when used to define products, compositions and methods,the term “comprising” (and any form of comprising, such as “comprise”and “comprises”), “having” (and any form of having, such as “have” and“has”), “including” (and any form of including, such as “includes” and“include”) or “containing” (and any form of containing, such as“contains” and “contain”) are open-ended and do not exclude additional,unrecited elements or method steps. Thus, a polypeptide “comprises” anamino acid sequence when the amino acid sequence might be part of thefinal amino acid sequence of the polypeptide. Such a polypeptide canhave up to several hundred additional amino acids residues. “Consistingessentially of” means excluding other components or steps of anyessential significance. Thus, a composition consisting essentially ofthe recited components would not exclude trace contaminants andpharmaceutically acceptable carriers. A polypeptide “consistsessentially of” an amino acid sequence when such an amino acid sequenceis present with eventually only a few additional amino acid residues.“Consisting of” means excluding more than trace elements of othercomponents or steps. For example, a polypeptide “consists of” an aminoacid sequence when the polypeptide does not contain any amino acids butthe recited amino acid sequence.

The terms “polypeptide”, “peptide” and “protein” refer to polymers ofamino acid residues which comprise at least nine or more amino acidsbonded via peptide bonds. The polymer can be linear, branched or cyclicand may comprise naturally occurring and/or amino acid analogs and itmay be interrupted by non-amino acids. As a general indication, if theamino acid polymer is more than 50 amino acid residues, it is preferablyreferred to as a polypeptide or a protein whereas if it is 50 aminoacids long or less, it is referred to as a “peptide”.

Within the context of the present invention, the terms “nucleic acid”,“nucleic acid molecule”, “polynucleotide” and “nucleotide sequence” areused interchangeably and define a polymer of any length of eitherpolydeoxyribonucleotides (DNA) (e.g. cDNA, genomic DNA, plasmids,vectors, viral genomes, isolated DNA, probes, primers and any mixturethereof) or polyribonucleotides (RNA) (e.g. mRNA, antisense RNA, SiRNA)or mixed polyribo-polydeoxyribonucleotides. They encompass single ordouble-stranded, linear or circular, natural or synthetic, modified orunmodified polynucleotides. Moreover, a polynucleotide may comprisenon-naturally occurring nucleotides and may be interrupted bynon-nucleotide components.

The term “analog” as used herein refers to a molecule (polypeptide ornucleic acid) exhibiting one or more modification(s) with respect to thenative counterpart. Any modification(s) can be envisaged, includingsubstitution, insertion and/or deletion of one or more nucleotide/aminoacid residue(s). Preferred are analogs that retain a degree of sequenceidentity of at least 80%, preferably at least 85%, more preferably atleast 90%, and even more preferably at least 98% identity with thesequence of the native counterpart.

In a general manner, the term “identity” refers to an amino acid toamino acid or nucleotide to nucleotide correspondence between twopolypeptide or nucleic acid sequences. The percentage of identitybetween two sequences is a function of the number of identical positionsshared by the sequences, taking into account the number of gaps whichneed to be introduced for optimal alignment and the length of each gap.Various computer programs and mathematical algorithms are available inthe art to determine the percentage of identity between amino acidsequences, such as for example the Blast program available at NCBI orALIGN in Atlas of Protein Sequence and Structure (Dayhoffed, 1981,Suppl., 3: 482-9). Programs for determining identity between nucleotidesequences are also available in specialized data base (e.g. Genbank, theWisconsin Sequence Analysis Package, BESTFIT, FASTA and GAP programs).For illustrative purposes, “at least 80% identity” means 80%, 81%, 82%,83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99% or 100%.

As used herein, the term “isolated” refers to a protein, polypeptide,peptide, polynucleotide, vector, etc., that is removed from its naturalenvironment (i.e. separated from at least one other component(s) withwhich it is naturally associated or found in nature). For example, anucleotide sequence is isolated when it is separated of sequencesnormally associated with it in nature (e.g. dissociated from a genome)but it can be associated with heterologous sequences.

The term “obtained from”, “originating” or “originate” is used toidentify the original source of a component (e.g. polypeptide, nucleicacid molecule) but is not meant to limit the method by which thecomponent is made which can be, for example, by chemical synthesis orrecombinant means.

As used herein, the term “host cell” should be understood broadlywithout any limitation concerning particular organization in tissue,organ, or isolated cells. Such cells may be of a unique type of cells ora group of different types of cells such as cultured cell lines, primarycells and dividing cells. In the context of the invention, the term“host cells” include prokaryotic cells, lower eukaryotic cells such asyeast, and other eukaryotic cells such as insect cells, plant andmammalian (e.g. human or non-human) cells as well as cells capable ofproducing the oncolytic virus and/or the immune checkpoint modulator(s)for use in the invention. This term also includes cells which can be orhas been the recipient of the vectors described herein as well asprogeny of such cells.

As used herein, the term “oncolytic virus” refers to a virus capable ofselectively replicating in dividing cells (e.g. a proliferative cellsuch as a cancer cell) with the aim of slowing the growth and/or lysingsaid dividing cell, either in vitro or in vivo, while showing no orminimal replication in non-dividing cells. Typically, an oncolytic viruscontains a viral genome packaged into a viral particle (or virion) andis infectious (i.e. capable of infecting and entering into a host cellor subject).

The term “treatment” (and any form of treatment such as “treating”,“treat”) as used herein encompasses prophylaxis (e.g. preventive measurein a subject at risk of having the pathological condition to be treated)and/or therapy (e.g. in a subject diagnosed as having the pathologicalcondition), eventually in association with conventional therapeuticmodalities. The result of the treatment is to slow down, cure,ameliorate or control the progression of the targeted pathologicalcondition. For example, a subject is successfully treated for a cancerif after administration of an oncolytic virus and of one or more immunecheck point modulator(s) as described herein, the subject shows anobservable improvement of its clinical status.

The term “administering” (or any form of administration such as“administered”) as used herein refers to the delivery to a subject of atherapeutic agent such as the oncolytic virus and/or the immunecheckpoint modulator(s) described herein.

As used herein, the term “proliferative disease” encompasses any diseaseor condition resulting from uncontrolled cell growth and spreadincluding cancers as well as diseases associated to an increasedosteoclast activity (e.g. rheumatoid arthritis, osteoporosis, etc) andcardiovascular diseases (restenosis that results from the proliferationof the smooth muscle cells of the blood vessel wall, etc). The term“cancer” may be used interchangeably with any of the terms “tumor”,“malignancy”, “neoplasm”, etc. These terms are meant to include any typeof tissue, organ or cell, any stage of malignancy (e.g. from a prelesionto stage IV)

The term “subject” generally refers to an organism for whom any productand method of the invention is needed or may be beneficial. Typically,the organism is a mammal, particularly a mammal selected from the groupconsisting of domestic animals, farm animals, sport animals, andprimates. Preferably, the subject is a human who has been diagnosed ashaving or at risk of having a proliferative disease such as a cancer.The terms “subject” and “patients” may be used interchangeably whenreferring to a human organism and encompasses male and female. Thesubject to be treated may be a newborn, an infant, a young adult or anadult.

The term “combination” as used herein refers to any arrangement possibleof various components (e.g. oncolytic virus and immune checkpointmodulator(s)). Such an arrangement includes mixture of at least oneoncolytic virus with one or more immune check point modulator(s) in theform of polypeptides (e.g. recombinant antibody or mixture ofrecombinant antibodies) or nucleic acid molecule(s) (e.g. carried by oneor more vector engineered for expressing such one or more immunecheckpoint modulator(s)) as well as mixture of polypeptide(s) andnucleic acid molecule(s) (e.g. a recombinant antibody and an expressingvector). The present invention encompasses combinations comprising equalmolar concentrations of each immune checkpoint modulator when more thanone is used as well as combinations with very different concentrations.It is appreciated that optimal concentration of each component of thecombination can be determined by the artisan skilled in the art.Preferably the combination is synergistic providing higher efficacy thaneach entity alone.

The term “immune checkpoint modulator” refers to a molecule capable ofmodulating the function of an immune checkpoint protein in a positive ornegative way (in particular the interaction between an antigenpresenting cell (APC) such as a cancer cell and an immune T effectorcell). The term “immune checkpoint” refers to a protein directly orindirectly involved in an immune pathway that under normal physiologicalconditions is crucial for preventing uncontrolled immune reactions andthus for the maintenance of self-tolerance and/or tissue protection. Theone or more immune checkpoint modulator(s) in use herein mayindependently act at any step of the T cell-mediated immunity includingclonal selection of antigen-specific cells, T cell activation,proliferation, trafficking to sites of antigen and inflammation,execution of direct effector function and signaling through cytokinesand membrane ligands. Each of these steps is regulated bycounterbalancing stimulatory and inhibitory signals that in fine tunethe response. In the context of the present invention, the termencompasses immune checkpoint modulator(s) capable of down-regulating atleast partially the function of an inhibitory immune checkpoint(antagonist) and/or immune checkpoint modulator(s) capable ofup-regulating at least partially the function of a stimulatory immunecheckpoint (agonist).

Oncolytic Virus

The oncolytic virus for use in the present invention can be obtainedfrom any member of virus identified at present time provided that it isoncolytic by its propensity to selectivity replicate and kill dividingcells as compared to non-dividing cells. It may be a native virus thatis naturally oncolytic or may be engineered by modifying one or moreviral genes so as to increase tumor selectivity and/or preferentialreplication in dividing cells, such as those involved in DNAreplication, nucleic acid metabolism, host tropism, surface attachment,virulence, lysis and spread (see for example Kirn et al., 2001, Nat.Med. 7: 781; Wong et al., 2010, Viruses 2: 78-106). One may alsoenvisage placing one or more viral gene(s) under the control of event ortissue-specific regulatory elements (e.g. promoter).

Exemplary oncolytic viruses include without limitation reovirus, SenecaValley virus (SVV), vesicular stomatitis virus (VSV), Newcastle diseasevirus (NDV), herpes simplex virus (HSV), morbillivirus virus,retrovirus, influenza virus, Sin bis virus, poxvirus, adenovirus, or thelike.

In one embodiment, the oncolytic virus for use in the present inventionis obtained from a reovirus. A representative example includes Reolysin(under development by Oncolytics Biotech; NCT01166542).

In one embodiment, the oncolytic virus for use in the present inventionis obtained from a Seneca Valley virus. A representative exampleincludes NTX-010 (Rudin et al., 2011, Clin. Cancer. Res. 17(4): 888-95).

In one embodiment, the oncolytic virus for use in the present inventionis obtained from a vesicular stomatitis virus (VSV). Representativeexamples for use in the invention are described in the literature (e.g.Stojdl et al., 2000, Nat. Med. 6(7): 821-5; Stojdl et al., 2003, CancerCell 4(4): 263-75).

In one embodiment, the oncolytic virus for use in the present inventionis obtained from a Newcastle disease virus. Representative examples foruse in the invention include without limitation the 73-T PV701 andHDV-HUJ strains as well as those described in the literature (e.g.Phuangsab et al., 2001, Cancer Lett. 172(1): 27-36; Lorence et al.,2007, Curr. Cancer Drug Targets 7(2): 157-67; Freeman et al., 2006, Mol.Ther. 13(1): 221-8).

In one embodiment, the oncolytic virus for use in the present inventionis obtained from a herpes simplex virus (HSV). The Herpesviridae are alarge family of DNA viruses that all share a common structure and arecomposed of relatively large double-stranded, linear DNA genomesencoding 100-200 genes encapsided within an icosahedral capsid which isenveloped in a lipid bilayer membrane. Although the oncolytic herpesvirus can be derived from different types of HSV, particularly preferredare HSV1 and HSV2. The herpes virus may be genetically modified so as torestrict viral replication in tumors or reduce its cytotoxicity innon-dividing cells. For example, any viral gene involved in nucleic acidmetabolism may be inactivated, such as thymidine kinase (Martuza et al.,1991, Science 252: 854-6), ribonucleotide reductase (RR) (Boviatsis etal., 1994, Gene Ther. 1: 323-31; Mineta et al., 1994, Cancer Res. 54:3363-66), or uracil-N-glycosylase (Pyles et al., 1994, J. Virol. 68:4963-72). Another aspect involves viral mutants with defects in thefunction of genes encoding virulence factors such as the ICP34.5 gene(Chambers et al., 1995, Proc. Natl. Acad. Sci. USA 92: 1411-5).Representative examples of oncolytic herpes virus include NV1020 (e.g.Geevarghese et al., 2010, Hum. Gene Ther. 21(9): 1119-28) and T-VEC(Andtbacka et al., 2013, J. Clin. Oncol. 31, abstract number LBA9008).

In one embodiment, the oncolytic virus for use in the present inventionis obtained from a morbillivirus which can be obtained from theparamyxoviridae family, with a specific preference for measles virus.Representative examples of suitable oncolytic measles viruses includewithout limitation MV-Edm (McDonald et al., 2006; Breast Cancer Treat.99(2): 177-84) and HMWMAA (Kaufmann et al., 2013, J. Invest. Dermatol.133(4): 1034-42)

In one embodiment, the oncolytic virus for use in the present inventionis obtained from an adenovirus. Methods are available in the art toengineer oncolytic adenoviruses. An advantageous strategy includes thereplacement of viral promoters with tumor-selective promoters ormodifications of the E1 adenoviral gene product(s) to inactivateits/their binding function with p53 or retinoblastoma (Rb) protein thatare altered in tumor cells. In the natural context, the adenovirus E1B55kDa gene cooperates with another adenoviral product to inactivate p53(p53 is frequently dysregulated in cancer cells), thus preventingapoptosis. Representative examples of oncolytic adenovirus includeONYX-015 (e.g. Khuri et al., 2000, Nat. Med 6(8): 879-85) and H101 alsonamed Oncorine (Xia et al., 2004, Ai Zheng 23(12): 1666-70).

In one embodiment, the oncolytic virus for use in the present inventionis a poxvirus. As used herein the term “poxvirus” refers to a virusbelonging to the Poxviridae family, with a specific preference for apoxvirus belonging to the Chordopoxviridae subfamily and more preferablyto the Orthopoxvirus genus. Sequences of the genome of variouspoxviruses, for example, the vaccinia virus, cowpox virus, Canarypoxvirus, Ectromelia virus, Myxoma virus genomes are available in the artand specialized databases such as Genbank (accession number NC_006998,NC_003663, NC_005309, NC_004105, NC_001132 respectively).

Desirably, the oncolytic poxvirus is an oncolytic vaccinia virus.Vaccinia viruses are members of the poxvirus family characterized by a200 kb double-stranded DNA genome that encodes numerous viral enzymesand factors that enable the virus to replicate independently from thehost cell machinery. The majority of vaccinia virus particles isintracellular (IMV for intracellular mature virion) with a single lipidenvelop and remains in the cytosol of infected cells until lysis. Theother infectious form is a double enveloped particle (EEV forextracellular enveloped virion) that buds out from the infected cellwithout lysing it.

Although it can derive from any vaccinia virus strain, Elstree, Wyeth,Copenhagen and Western Reserve strains are particularly preferred. Thegene nomenclature used herein is that of Copenhagen vaccinia strain. Itis also used herein for the homologous genes of other poxviridae unlessotherwise indicated. However, gene nomenclature may be differentaccording to the pox strain but correspondence between Copenhagen andother vaccinia strains are generally available in the literature.

Preferably, the oncolytic vaccinia virus for use in the presentinvention is modified by altering one or more viral gene(s). Saidmodification(s) preferably lead(s) to the synthesis (or lack ofsynthesis) of a defective protein unable to ensure the activity of theprotein produced under normal conditions by the unmodified gene.Modifications encompass deletion, mutation and/or substitution of one ormore nucleotide(s) (contiguous or not) within the viral gene or itsregulatory elements. Modification(s) can be made in a number of waysknown to those skilled in the art using conventional recombinanttechniques. Exemplary modifications are disclosed in the literature witha specific preference for those altering viral genes involved in DNAmetabolism, host virulence and IFN pathway (see e.g. Guse et al., 2011,Expert Opinion Biol. Ther. 11(5):595-608).

More preferably, the oncolytic poxvirus for use in the present inventionis modified by altering the thymidine kinase-encoding gene (locus J2R).The TK enzyme is involved in the synthesis of deoxyribonucleotides. TKis needed for viral replication in normal cells as these cells havegenerally low concentration of nucleotides whereas it is dispensable individing cells which contain high nucleotide concentration.

Alternatively or in combination, the oncolytic poxvirus for use in thepresent invention is modified by altering at least one gene or bothgenes encoding Ribonucleotide reductase (RR). In the natural context,this enzyme catalyzes the reduction of ribonucleotides todeoxyribonucleotides that represents a crucial step in DNA biosynthesis.The viral enzyme is similar in subunit structure to the mammalianenzyme, being composed of two heterologous subunits, designed R1 and R2encoded respectively by the 14L and F4L locus. Sequences for the 14L andF4L genes and their locations in the genome of various poxvirus areavailable in public databases, for example via accession numberDQ437594, DQ437593, DQ377804, AH015635, AY313847, AY313848, NC_003391,NC_003389, NC_003310, M-35027, AY243312, DQ011157, DQ011156, DQ011155,DQ011154, DQ011153, Y16780, X71982, AF438165, U60315, AF410153,AF380138, U86916, L22579, NC_006998, DQ121394 and NC_008291. In thecontext of the invention, either the 14L gene (encoding the R1 largesubunit) or the F4L gene (encoding the R2 small subunit) or both may beinactivated.

Alternatively or in combination, other strategies may also be pursued tofurther increase the virus tumor-specificity. A representative exampleof suitable modifications includes disruption of the VGF-encoding genefrom the viral genome. VGF (for VV growth factor) is a secreted proteinwhich is expressed early after cell infection and its function seemsimportant for virus spread in normal cells. Another example is thedisruption of the A56R gene coding for hemagglutinin, eventually incombination with TK deletion (Zhang et al., 2007, Cancer Res. 67:10038-46). Disruption of interferon modulating gene(s) may also beadvantageous (e.g. the B8R or B18R gene) or the caspase-1 inhibitor B13Rgene.

In a preferred embodiment, the oncolytic virus for use in this inventionis a vaccinia virus defective for TK resulting from inactivatingmutations in the J2R gene. In another preferred embodiment, theoncolytic virus for use in this invention is a vaccinia virus defectivefor both TK and RR activities resulting from inactivating mutations inboth the J2R gene and the 14L and/or F4L gene(s) carried by the viralgenome (e.g. as described in WO2009/065546 and Foloppe et al., 2008,Gene Ther., 15: 1361-71).

Therapeutic Genes

In one embodiment, the oncolytic virus for use in this invention furtherexpresses at least one therapeutic gene inserted in the viral genome. A“therapeutic gene” encodes a product capable of providing a biologicalactivity when administered appropriately to a subject, which is expectedto cause a beneficial effect on the course or a symptom of thepathological condition to be treated by either potentiating anti-tumorefficacy or reinforcing the oncolytic nature of the virus. In thecontext of the invention, the therapeutic gene can be of human origin ornot (e.g. of bacterial, yeast or viral origin). Preferably, thetherapeutic gene is not a gene or nucleic acid sequence encoding animmune checkpoint modulator as described herein.

A vast number of therapeutic genes may be envisaged in the context ofthe invention such as those encoding polypeptides that can compensatefor defective or deficient proteins in the subject, or those that actthrough toxic effects to limit or remove harmful cells from the body orthose that encode immunity conferring polypeptides. They may be nativegenes or genes obtained from the latter by mutation, deletion,substitution and/or addition of one or more nucleotides.

Advantageously, the oncolytic virus in use in the present inventioncarries a therapeutic gene selected from the group consisting of genesencoding suicide gene products and immunostimulatory proteins.

Suicide Gene

The term “suicide gene” refers to a gene coding for a protein able toconvert a precursor of a drug into a cytoxic compound. Suicide genescomprise but are not limited to genes coding protein having a cytosinedeaminase activity, a thymidine kinase activity, an uracilphosphoribosyl transferase activity, a purine nucleoside phosphorylaseactivity and a thymidylate kinase activity. Examples of suicide genesand corresponding precursors of a drug comprising one nucleobase moietyare disclosed in the following table

TABLE 1 Suicide gene prodrug Thymidine Kinase Ganciclovir; Ganciclovirelaidic acid ester; penciclovir; Acyclovir; Valacyclovir; (E)-5-(2-bromovinyl)-2′-deoxyuridine; zidovudine; 2′- Exo-methanocarbathymidineCytosine deaminase 5-Fluorocytosine Purine nucleoside 6-Methylpurinedeoxyriboside; phosphorylase Fludarabine uracil phosphoribosyl5-Fluorocytosine; 5-Fluorouracil transferase thymidylate kinase.Azidothymidine

Desirably, the suicide gene encodes a protein having at least cytosinedeaminase (CDase) activity. In the prokaryotes and lower eukaryotes (itis not present in mammals), CDase is involved in the pyrimidinemetabolic pathway by which exogenous cytosine is transformed into uracilby means of a hydrolytic deamination. CDase also deaminates an analogueof cytosine, i.e. 5-fluorocytosine (5-FC), thereby forming5-fluorouracil (5-FU), a compound which is highly cytotoxic when it isconverted into 5-fluoro-UMP (5-FUMP). CDase encoding nucleic acidmolecule can be obtained from any prokaryotes and lower eukaryotes suchas Saccharomyces cerevisiae (FCY1 gene), Candida Albicans (FCA1 gene)and Escherichia coli (codA gene). The gene sequences and encoded CDaseproteins have been published and are available in specialized data banks(SWISSPROT EMBL, Genbank, Medline and the like). Functional analogues ofthese genes may also be used. Such analogues preferably have a nucleicacid sequence having a degree of identity of at least 70%,advantageously of at least 80%, preferably of at least 90%, and mostpreferably of at least 95% with the nucleic acid sequence of the nativegene.

Alternatively or in combination, the oncolytic virus in use in theinvention carries in its viral genome a suicide gene encoding apolypeptide having uracil phosphoribosyl transferase (UPRTase) activity.In prokaryotes and lower eukaryotes, uracil is transformed into UMP bythe action of UPRTase. This enzyme converts 5-FU into 5-FUMP. By way ofillustration, the nucleic acid sequences encoding the UPRTases from E.coli (Andersen et al., 1992, European J. Biochem. 204: 51-56), fromLactococcus lactis (Martinussen et al., 1994, J. Bacteriol. 176:6457-63), from Mycobacterium bovis (Kim et al., 1997, Biochem. Mol.Biol. Internat. 41: 1117-24) and from Bacillus subtilis (Martinussen etal., 1995, J. Bacteriol. 177: 271-4) may be used in the context of theinvention. However, it is most particularly preferred to use a yeastUPRTase and in particular that encoded by the S. cerevisiae (FUR1 gene)whose sequence is disclosed in Kern et al. (1990, Gene 88: 149-57).Functional UPRTase analogues may also be used such as the N-terminallytruncated FUR1 mutant described in EP998568 (with a deletion of the 35first residues up to the second Met residue present at position 36 inthe native protein) which exhibits a higher UPRTase activity than thatof the native enzyme.

Preferably, the suicide gene inserted in the viral genome of theoncolytic virus for use in the present invention encodes a polypeptidehaving CDase and UPRTase activities. Such a polypeptide can beengineered by fusion of two enzymatic domains, one having the CDaseactivity and the second having the UPRTase activity. Exemplarypolypeptides include without limitation fusion polypeptides codA::upp,FCY1::FUR1 and FCY1::FUR1[Delta] 105 (FCU1) and FCU1-8 described inWO96/16183, EP998568 and WO2005/07857. Of particular interest is theFCU1 suicide gene (or FCY1::FUR1[Delta] 105 fusion) encoding apolypeptide comprising the amino acid sequence represented in thesequence identifier SEQ ID NO: 1 of WO2009/065546. The present inventionencompasses analogs of such polypeptides providing they retain theCDase, and/or UPRTase activities. It is within the reach of the skilledperson to isolate the CDase and/or UPRTase-encoding nucleic acidmolecules from the published data, eventually engineer analogs thereofand test the enzymatic activity in an acellular or cellular systemaccording to conventional techniques (see e.g. EP998568).

Immunostimulatory Therapeutic Genes

As used herein, the term “immunostimulatory protein” refers to a proteinwhich has the ability to stimulate the immune system, in a specific ornon-specific way. A vast number of proteins are known in the art fortheir ability to exert an immunostimulatory effect. Examples of suitableimmunostimulatory proteins in the context of the invention includewithout limitation cytokines, with a specific preference forinterleukins (e.g. IL-2, IL-6, IL-12, IL-15, IL-24), chemokines (e.g.CXCL10, CXCL9, CXCL11), interferons (e.g. IFNg, IFNalpha), tumornecrosis factor (TNF), colony-stimulating factors (e.g. GM-CSF, C-CSF,M-CSF . . . ), APC (for Antigen Presenting Cell)-exposed proteins (e.g.B7.1, B7.2 and the like), growth factors (Transforming Growth FactorTGF, Fibroblast Growth Factor FGF, Vascular Endothelial Growth FactorsVEGF, and the like), major histocompatibility complex (MHC) antigens ofclass I or II, apoptosis inducers or inhibitors (e.g. Bax, BcI2, BcIX .. . ), cytostatic agents (p21, p16, Rb . . . ), immunotoxins, antigens(antigenic polypeptides, epitopes, and the like) and markers(beta-galactosidase, luciferase . . . ). Preferably, theimunostimulatory protein is an interleukin or a colony-stimulatingfactor, with a specific preference for GM-CSF.

Expression of the Therapeutic Genes

The therapeutic gene may be easily obtained by cloning, by PCR or bychemical synthesis according to the conventional techniques. Inaddition, the therapeutic gene can be optimized for providing high levelexpression in a particular host cell or subject. It has been indeedobserved that, the codon usage patterns of organisms are highlynon-random and the use of codons may be markedly different betweendifferent hosts. As the therapeutic gene might be from bacterial orlower eukaryote origin (e.g. the suicide gene), it may have aninappropriate codon usage pattern for efficient expression in highereukaryotic cells (e.g. human). Typically, codon optimization isperformed by replacing one or more “native” (e.g. bacterial or yeast)codon corresponding to a codon infrequently used in the host organism ofinterest by one or more codon encoding the same amino acid which is morefrequently used. It is not necessary to replace all native codonscorresponding to infrequently used codons since increased expression canbe achieved even with partial replacement.

Further to optimization of the codon usage, expression in the host cellor subject can further be improved through additional modifications ofthe gene sequence. For example, the therapeutic gene sequence can bemodified so as to prevent clustering of rare, non-optimal codons beingpresent in concentrated areas and/or to suppress or modify “negative”sequence elements which are expected to negatively influence expressionlevels. Such negative sequence elements include without limitation theregions having very high (>80%) or very low (<30%) GC content; AT-richor GC-rich sequence stretches; unstable direct or inverted repeatsequences; R A secondary structures; and/or internal cryptic regulatoryelements such as internal TATA-boxes, chi-sites, ribosome entry sites,and/or splicing donor/acceptor sites.

In accordance with the present invention, the therapeutic gene(s)inserted in the genome of the oncolytic virus for use in the inventionis operably linked to suitable regulatory elements for its expression ina host cell or subject. As used herein, the term “regulatory elements”or “regulatory sequence” refers to any element that allows, contributesor modulates the expression of the therapeutic gene(s) in a given hostcell or subject, including replication, duplication, transcription,splicing, translation, stability and/or transport of the nucleic acid(s)or its derivative (i.e. m RNA). As used herein, “operably linked” meansthat the elements being linked are arranged so that they function inconcert for their intended purposes. For example a promoter is operablylinked to a nucleic acid molecule if the promoter effects transcriptionfrom the transcription initiation to the terminator of said nucleic acidmolecule in a permissive host cell.

It will be appreciated by those skilled in the art that the choice ofthe regulatory sequences can depend on such factors as the gene itself,the virus into which it is inserted, the host cell or subject, the levelof expression desired, etc. The promoter is of special importance. Inthe context of the invention, it can be constitutive directingexpression of the therapeutic gene(s) in many types of host cells orspecific to certain host cells (e.g. liver-specific regulatorysequences) or regulated in response to specific events or exogenousfactors (e.g. by temperature, nutrient additive, hormone, etc) oraccording to the phase of a viral cycle (e.g. late or early). One mayalso use promoters that are repressed during the production step inresponse to specific events or exogenous factors, in order to optimizevirus production and circumvent potential toxicity of the expressedpolypeptide(s).

Promoters suitable for constitutive expression in mammalian cellsinclude but are not limited to the cytomegalovirus (CMV) immediate earlypromoter (U.S. Pat. No. 5,168,062), the RSV promoter, the adenovirusmajor late promoter, the phosphoglycero kinase (PGK) promoter (Adra etal., 1987, Gene 60: 65-74), the thymidine kinase (TK) promoter of herpessimplex virus (HSV)-1 and the T7 polymerase promoter (WO98/10088).Vaccinia virus promoters are particularly adapted for expression inoncolytic poxviruses. Representative examples include without limitationthe vaccinia 7.5K, HSR, 11K7.5 (Erbs et al., 2008, Cancer Gene Ther.15(1): 18-28), TK, p28, p11 and K1L promoter, as well as syntheticpromoters such as those described in Chakrabarti et al. (1997,Biotechniques 23: 1094-7; Hammond et al, 1997, J. Virol Methods 66:135-8; and Kumar and Boyle, 1990, Virology 179: 151-8) as well asearly/late chimeric promoters. Promoters suitable for oncolytic measlesviruses include without limitation any promoter directing expression ofmeasles transcription units (Brandler and Tangy, 2008, CIMID 31: 271).

Those skilled in the art will appreciate that the regulatory elementscontrolling the expression of the therapeutic gene(s) may furthercomprise additional elements for proper initiation, regulation and/ortermination of transcription (e.g. polyA transcription terminationsequences), mRNA transport (e.g. nuclear localization signal sequences),processing (e.g. splicing signals), and stability (e.g. introns andnon-coding 5′ and 3′ sequences), translation (e.g. an initiator Met,tripartite leader sequences, IRES ribosome binding sites, signalpeptides, etc.).

The therapeutic gene can be inserted at any location of the viralgenome, with a specific preference for a non-essential locus Forexample, TK gene is particularly appropriate for insertion in oncolyticvaccinia virus.

In a preferred embodiment, the oncolytic virus for use in the inventionis a vaccinia virus (preferably from the Copenhague strain) defectivefor both TK and RR activities (e.g. resulting from inactivatingmutations in both the viral J2R and 14L genes). More preferably, saidvaccinia virus is armed with a suicide gene with a special preferencefor the FCU1 suicide gene described herein. Even more preferably, thesuicide gene (e.g. FCU1) is under the transcriptional control of thep11K7.5 vaccinia promoter. Still more preferably, the FCU1 placed underthe control of the vaccinia virus promoter is inserted within TK locusof the virus genome.

In an alternative and also preferred embodiment, the oncolytic virus foruse in the invention is a vaccinia virus (preferably from the Wyethstrain) defective for TK activity (resulting from inactivating mutationsin the virus J2R gene). More preferably, said vaccinia virus is armedwith an immunostimulatory therapeutic gene with a special preference forthe human GM-CSF gene described herein. Even more preferably, thetherapeutic gene (e.g. GM-CSF) is under the transcriptional control of asynthetic early-late promoter vaccinia promoter and is preferablyinserted within TK locus.

Typically, the oncolytic virus for use according to the presentinvention is produced into a suitable host cell line using conventionaltechniques including culturing the transfected or infected host cellunder suitable conditions so as to allow the production of infectiousviral particles and recovering the produced infectious viral particlesfrom the culture of said cell and optionally purifying said recoveredinfectious viral particles. Suitable host cells for production of theoncolytic virus include without limitation human cell lines such as HeLa(ATCC), 293 cells (Graham et al., 1997, J. Gen. Virol. 36: 59-72),HER96, PER-C6 (Fallaux et al., 1998, Human Gene Ther. 9: 1909-17), aviancells such as those described in WO2005/042728, WO2006/108846,WO2008/129058, WO2010/130756, WO2012/001075, etc), hamster cell linessuch as BHK-21 (ATCC CCL-10) as well as primary chicken embryofibroblasts (CEF) prepared from chicken embryos obtained from fertilizedeggs. The oncolytic virus can be at least partially isolated beforebeing used according to the present invention. Various purificationsteps can be envisaged, including clarification, enzymatic treatment(e.g. benzonase, protease), chromatographic and filtration steps.Appropriate methods are described in the art (e.g. WO2007/147528;WO2008/138533, WO2009/100521, WO2010/130753, WO2013/022764).

Immune Checkpoint Modulator(s)

Immune checkpoints and modulators thereof as well as methods of usingsuch compounds are described in the literature. In accordance with thisinvention, the one or more immune checkpoint modulator(s) mayindependently be a polypeptide or a polypeptide-encoding nucleic acidmolecule; said polypeptide comprising a domain capable of binding thetargeted immune checkpoint and/or inhibiting the binding of a ligand tosaid targeted immune checkpoint so as to exert an antagonist function(i.e. being capable of antagonizing an immune checkpoint-mediatedinhibitory signal) or an agonist function (i.e. being capable ofboosting an immune checkpoint-mediated stimulatory signal). Such one ormore immune checkpoint modulator(s) can be independently selected fromthe group consisting of peptides (e.g. peptide ligands), soluble domainsof natural receptors, RNAi, antisense molecules, antibodies and proteinscaffolds.

In a preferred embodiment, the immune checkpoint modulator is anantibody. In the context of the invention, “antibody” (“Ab”) is used inthe broadest sense and encompasses naturally occurring and engineered byman as well as full length antibodies or functional fragments or analogsthereof that are capable of binding the target immune checkpoint orepitope (thus retaining the target-binding portion). The antibody in usein the invention can be of any origin, e.g. human, humanized, animal(e.g. rodent or camelid antibody) or chimeric. It may be of any isotypewith a specific preference for an IgG1 or IgG4 isotype. In addition, itmay be glycosylated or non-glycosylated. The term antibody also includesbispecific or multispecific antibodies so long as they exhibit thebinding specificity described herein.

For illustrative purposes, full length antibodies are glycoproteinscomprising at least two heavy (H) chains and two light (L) chainsinter-connected by disulfide bonds. Each heavy chain is comprised of aheavy chain variable region (VH) and a heavy chain constant region whichis made of three CH1, CH2 and CH3 domains (eventually with a hingebetween CH1 and CH2). Each light chain is comprised of a light chainvariable region (VL) and a light chain constant region which comprisesone CL domain. The VH and VL regions comprise hypervariable regions,named complementarity determining regions (CDR), interspersed with moreconserved regions named framework regions (FR). Each VH and VL iscomposed of three CDRs and four FRs in the following order:FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. The CDR regions of the heavy and lightchains are determinant for the binding specificity.

As used herein, an “humanized antibody” refers to a non-human (e.g.murine, camel, rat, etc) antibody whose protein sequence has beenmodified to increase its similarity to a human antibody (i.e. producednaturally in humans). The process of humanization is well known in theart (see e.g. Presta et al., 1997, Cancer Res. 57(20): 4593-9; U.S. Pat.Nos. 5,225,539; 5,530,101; 6,180,370; WO2012/110360). For example, amonoclonal antibody developed for human use can be humanized bysubstituting one or more residue of the FR regions to look like humanimmunoglobulin sequence whereas the vast majority of the residues of thevariable regions (especially the CDRs) are not modified and correspondto those of a non-human immunoglobulin. For general guidance, the numberof these amino acid substitutions in the FR regions is typically no morethan 20 in each variable region VH or VL.

As used herein, a “chimeric antibody” refers to an antibody comprisingone or more element(s) of one species and one or more element(s) ofanother species, for example, a non-human antibody comprising at least aportion of a constant region (Fc) of a human immunoglobulin.

Many forms of antibody can be engineered for use in the combination ofthe invention. Representative examples include without limitation Fab,Fab′, F(ab′)2, dAb, Fd, Fv, scFv, di-scFv and diabody, etc. Morespecifically:

-   -   (i) a Fab fragment represented by a monovalent fragment        consisting of the VL, VH, CL and CH1 domains;    -   (ii) a F(ab′)2 fragment represented by a bivalent fragment        comprising two Fab fragments linked by at least one disulfide        bridge at the hinge region;    -   (iii) a Fd fragment consisting of the VH and CH1 domains;    -   (iv) a Fv fragment consisting of the VL and VH domains of a        single arm of an antibody,    -   (v) a dAb fragment consisting of a single variable domain        fragment (VH or VL domain);    -   (vi) a single chain Fv (scFv) comprising the two domains of a Fv        fragment, VL and VH, that are fused together, eventually with a        linker to make a single protein chain (see e.g. Bird et al.,        1988, Science 242: 423-6; Huston et al., 1988, Proc. Natl. Acad.        Sci. USA 85: 5879-83; U.S. Pat. Nos. 4,946,778; 5,258,498); and    -   (vii) any other artificial antibody.

Methods for preparing antibodies, fragments and analogs thereof areknown in the art (see e.g. Harlow and Lane, 1988, Antibodies—Alaboratory manual; Cold Spring Harbor Laboratory, Cold Spring HarborN.Y.). One may cite for example hybridoma technology (as described inKohler and Milstein, 1975, Nature 256: 495-7; Cote et al., 1983, Proc.Natl. Acad. Sci. USA 80: 2026-30; Cole et al. in Monoclonal antibodiesand Cancer Therapy; Alan Liss pp 77-96), recombinant techniques (e.g.using phage display methods), peptide synthesis and enzymatic cleavage.Antibody fragments can be produced by recombinant technique as describedherein. They may also be produced by proteolytic cleavage with enzymessuch as papain to produce Fab fragments or pepsin to produce F(ab′)2fragments as described in the literature (see e.g. Wahl et al., 1983, J.Nucl. Med. 24: 316-25). Analogs (or fragment thereof) can be generatedby conventional molecular biology methods (PCR, mutagenesis techniques).If needed, such fragments and analogs may be screened for functionalityin the same manner as with intact antibodies (e.g. by standard ELISAassay).

In a preferred embodiment, at least one of the one or more immunecheckpoint modulator(s) for use in the present invention is a monoclonalantibody, with a specific preference for a human (in which both theframework regions are derived from human germline immunoglobinsequences) or a humanized antibody according to well-known humanizationprocess.

Desirably, the one or more immune checkpoint modulator(s) in use in thepresent invention antagonizes at least partially (e.g. more than 50%)the activity of inhibitory immune checkpoint(s), in particular thosemediated by any of the following PD-1, PD-L1, PD-L2, LAG3, Tim3, KIR,BTLA and CTLA4, with a specific preference for a monoclonal antibodythat specifically binds to any of such target proteins. The term“specifically binds to” refers to the capacity to a binding specificityand affinity for a particular target or epitope even in the presence ofa heterogeneous population of other proteins and biologics. Thus, underdesignated assay conditions, the antibody in use in the invention bindspreferentially to its target and does not bind in a significant amountto other components present in a test sample or subject. Preferably,such an antibody shows high affinity binding to its target with anequilibrium dissociation constant equal or below 1×10⁻⁶M (e.g. at least0.5×10⁻⁶, 1×10⁻⁷, 1×10⁻⁸, 1×10⁻⁹, 1×10⁻¹⁰, etc). Alternatively, the oneor more immune checkpoint modulator(s) in use in the present inventionexerts an agonist function in the sense that it is capable ofstimulating or reinforcing stimulatory signals, in particular thosemediated by CD28 with a specific preference for any of ICOS, CD137 (or4-1BB), OX40, CD27, CD40 and GITR immune checkpoints. Standard assays toevaluate the binding ability of the antibodies toward immune checkpointsare known in the art, including for example, ELISAs, Western blots, RIAsand flow cytometry. The binding kinetics (e.g., binding affinity) of theantibodies also can be assessed by standard assays known in the art,such as by Biacore analysis.

In a preferred embodiment, at least one of the one or more checkpointmodulator(s) for use in this invention comprises a human or a humanizedantibody capable of antagonizing an immune checkpoint involved in Tcell-mediated response. A preferred example of immune checkpointmodulator is represented by a modulator capable of antagonizing at leastpartially the protein Programmed Death 1 (PD-1), and especially anantibody that specifically binds to human PD-1. PD-1 is part of theimmunoglobulin (Ig) gene superfamily and a member of the CD28 family. Itis a 55 kDa type 1 transmembrane protein expressed onantigen-experienced cells (e.g. activated B cells, T cells, and myeloidcells) (Agata et al., 1996, Int. Immunol. 8: 765-72; Okazaki et al.,2002, Curr. Opin. Immunol. 14: 391779-82; Bennett et al., 2003, J.Immunol 170: 711-8). In normal context, it acts by limiting the activityof T cells at the time of inflammatory response, thereby protectingnormal tissues from destruction (Topalian, 2012, Curr. Opin. Immunol.24: 207-12). Two ligands have been identified for PD-1, respectivelyPD-L1 (programmed death ligand 1) and PD-L2 (programmed death ligand 2)(Freeman et al., 2000, J. Exp. Med. 192: 1027-34; Carter et al., 2002,Eur. J. Immunol. 32: 634-43). PD-L1 was identified in 20-50% of humancancers (Dong et al., 2002, Nat. Med. 8: 787-9). The interaction betweenPD-1 and PD-L1 resulted in a decrease in tumor infiltrating lymphocytes,a decrease in T-cell receptor mediated proliferation, and immune evasionby the cancerous cells (Dong et al., 2003, J. Mol. Med. 81: 281-7; Blanket al., 2005, Cancer Immunol. Immunother. 54: 307-314). The completenucleotide and amino acid PD-1 sequences can be found under GenBankAccession No U64863 and NP_005009.2. A number of anti PD1 antibodies areavailable in the art (see e.g. those described in WO2004/004771;WO2004/056875; WO2006/121168; WO2008/156712; WO2009/014708;WO2009/114335; WO2013/043569; and WO2014/047350). Preferred anti PD-1antibodies in the context of this invention are FDA approved or underadvanced clinical development and one may use in particular an anti-PD-1antibody selected from the group consisting of Nivolumab (also termedBMS-936558 under development by Bristol Myer Squibb), Pembrolizumab(also termed Lanbrolizumab or MK-3475; under development by Merck), andPidilizumab (also termed CT-011 under development by CureTech).

Another preferred example of immune checkpoint modulator is representedby a modulator capable of antagonizing at least partially the PD-1ligand termed PD-L1, and especially an antibody that recognizes humanPD-L1. A number of anti PD-L1 antibodies are available in the art (seee.g. those described in EP1907000). Preferred anti PD-L1 antibodies areFDA approved or under advanced clinical development (e.g. MPDL3280Aunder development by Genentech/Roche and BMS-936559 under development byBristol Myer Squibb).

Still another preferred example of immune checkpoint modulator isrepresented by a modulator capable of antagonizing at least partiallythe CTLA-4 protein, and especially an antibody that recognizes humanCTLA-4. CTLA4 (for cytotoxic T-lymphocyte-associated antigen 4) alsoknown as CD152 was identified in 1987 (Brunet et al., 1987, Nature 328:267-70) and is encoded by the CTLA4 gene (Dariavach et al., Eur. J.Immunol. 18: 1901-5). CTLA4 is a member of the immunoglobulinsuperfamily of receptors. It is expressed on the surface of helper Tcells where it primarily regulates the amplitude of the early stages ofT cell activation. Recent work has suggested that CTLA-4 may function invivo by capturing and removing B7-1 and B7-2 from the membranes ofantigen-presenting cells, thus making these unavailable for triggeringof CD28 (Qureshi et al., Science, 2011, 332: 600-3). The complete CTLA-4nucleic acid sequence can be found under GenBank Accession No LI 5006. Anumber of anti CTLA-4 antibodies are available in the art (see e.g.those described in U.S. Pat. No. 8,491,895). Preferred anti CTLA-4antibodies in the context of this invention are FDA approved or underadvanced clinical development. One may cite more particularly ipilimumabmarketed by Bristol Myer Squibb as Yervoy (see e.g. U.S. Pat. Nos.6,984,720; 8,017,114), tremelimumab under development by—Pfizer (seee.g. U.S. Pat. Nos. 7,109,003 and 8,143,379) and single chain anti-CTLA4antibodies (see e.g. WO97/20574 and WO2007/123737).

Immune checkpoint modulator for antagonizing the LAG3 receptor may alsobe used in the combination of the present invention (see e.g. U.S. Pat.No. 5,773,578).

Another example of immune checkpoint modulator is represented by an OX40agonist such as agonist ligand of OX40 (OX40L) (see e.g. U.S. Pat. Nos.5,457,035, 7,622,444; WO03/082919) or an antibody directed to the OX40receptor (see e.g. U.S. Pat. No. 7,291,331 and WO03/106498).

Other examples of immune checkpoint modulators are represented byanti-KIR or anti-CD96 antibody targeting the inhibitory receptorsharboured by CD8+ T cells and NK cells.

The present invention encompasses a combination comprising more than oneimmune checkpoint modulator. A preferred example includes withoutlimitation using an anti-CTLA-4 antibody and an anti-PD-1 antibody incombination with an oncolytic virus as described herein.

Production of Immune Checkpoint Modulator

The one or more immune checkpoint modulator(s) for use in this inventioncan be produced by recombinant means using suitable expression vectorsand host cells.

Nucleic acid molecules encoding the relevant portion(s) of the desiredimmune checkpoint modulator can be obtained using standard molecularbiology techniques (e.g. PCR amplification, cDNA cloning, chemicalsynthesis) using sequence data accessible in the art and the informationprovided herein. For example, cDNAs encoding the light and heavy chainsof the antibody or their CDRs can be isolated from the producinghybridoma, immunoglobulin gene libraries or any available source.

In one embodiment, the nucleic acid molecule(s) encoding the immunecheckpoint modulator(s) can be cloned in a suitable vector and expressedin a host cell to produce said immune checkpoint modulator byrecombinant means. Insertion into the expression vector can be performedby routine molecular biology, e.g. as described in Sambrook et al.(2001, Molecular Cloning-A Laboratory Manual, Cold Spring HarborLaboratory). Insertion into an adenoviral vector or a poxviral vectorcan be performed through homologous recombination as describedrespectively in Chartier et al. (1996, J. Virol. 70: 4805-10) and Paulet al. (2002, Cancer gene Ther. 9: 470-7). As described herein inconnection with the therapeutic gene, the nucleic acid molecule(s)encoding the immune checkpoint modulator(s) may also be optimized forincreasing expression levels.

A variety of host-vector systems may be used or constructed to expressthe one or more immune checkpoint modulator(s) for use in the presentinvention, including prokaryotic organisms such as bacteria (e.g. E.coli or Bacillus subtilis); yeast (e.g. Saccharomyces cerevisiae,Saccharomyces pombe, Pichia pastoris); insect cell systems (e.g. Sf 9cells and baculovirus); plant cell systems (e.g. cauliflower mosaicvirus CaMV; tobacco mosaic virus TMV) and mammalian cell systems (e.g.cultured cells). Typically, such vectors are commercially available(e.g. in Invitrogen, Stratagene, Amersham Biosciences, Promega, etc.) oravailable from depositary institutions such as the American Type CultureCollection (ATCC, Rockville, Md.) or have been the subject of numerouspublications describing their sequence, organization and methods ofproducing, allowing the artisan to apply them.

Suitable vectors for use in prokaryotic systems include withoutlimitation pBR322 (Gibco BRL), pUC (Gibco BRL), pbluescript(Stratagene), p Poly (Lathe et al., 1987, Gene 57: 193-201), pTrc (Amannet al., 1988, Gene 69: 301-15); pET lid (Studier et al., 1990, GeneExpression Technology: Methods in Enzymology 185: 60-89); pIN (Inouye etal., 1985, Nucleic Acids Res. 13: 3101-9; Van Heeke et al., 1989, J.Biol. Chem. 264: 5503-9); and pGEX vectors where the nucleic acidmolecule can be expressed in fusion with glutathione S-transferase (GST)(Amersham Biosciences Product).

Suitable vectors for expression in yeast (e.g. S. cerevisiae) include,but are not limited to pYepSecl (Baldari et al., 1987, EMBO J. 6:229-34), pMFa (Kujan et al., 1982, Cell 30: 933-43), pJRY88 (Schultz etal., 1987, Gene 54: 113-23), pYES2 (Invitrogen Corporation) and pTEF-MF(Dualsystems Biotech Product).

Suitable plasmid vectors for expression in mammalian host cells include,without limitation, pREP4, pCEP4 (Invitrogene), pCI (Promega), pCDM8(Seed, 1987, Nature 329: 840) and pMT2PC (Kaufman et al., 1987, EMBO J.6: 187-95), pVAX and pgWiz (Gene Therapy System Inc; Himoudi et al.,2002, J. Virol. 76: 12735-46).

Viral-based expression systems can also be utilized in the context ofthe invention derived from a variety of different viruses (e.g.baculovirus, retrovirus, adenovirus, AAV, poxvirus, measles virus, andthe like). As used herein, the term “viral vector” encompasses vectorDNA as well as viral particles generated thereof. Viral vectors arepreferably replication-defective or replication-impaired.

Moreover, the expression vector used in the context of the presentinvention may also comprise one or more additional element(s) enablingmaintenance, propagation or expression of the nucleic acid moleculeencoding the immune checkpoint modulator in the host cell. Suchadditional elements include without limitation marker gene(s) in orderto facilitate identification and isolation of the recombinant host cells(e.g. by complementation of a cell auxotrophy or by antibioticresistance), stabilising elements (e.g. cer sequence as described inSummers and Sherrat, 1984, Cell 36: 1097-103 and DAP system as describedin U.S. Pat. No. 5,198,343), and integrative elements (e.g. LTR viralsequences and transposons).

Suitable marker genes for expression in prokaryotic host cells includetetracycline and ampicillin-resistance genes. Also, resistance genes canbe used for expression in mammalian host cells such as dihydrofolatereductase (dhfr) which confers resistance to methotrexate (Wigler etal., 1980, Proc. Natl. Acad. Sci. USA 77: 3567; O'Hare et al., 1981,Proc. Natl. Acad. Sci. USA 78: 1527); gpt which confers resistance tomycophenolic acid (Mulligan and Berg, 1981, Proc. Natl. Acad. Sci. USA78: 2072); neo which confers resistance to the aminoglycoside G-418(Colberre-Garapin et al., 1981, J. Mol. Biol. 150: 1); zeo which confersresistance to zeomycin, kana which confers resistance to kanamycin andhygro, which confers resistance to hygromycin (Santerre et al., 1984,Gene 30: 147). URA3 and LEU2 genes can be used for expression in yeastsystems, which provide for complementation of ura3 or leu2 yeastmutants.

The expression vector can, where appropriate, be combined with one ormore substances which improve the transfectional efficiency and/orstability of the vector. These substances are widely documented in theliterature. Representative examples of transfection reagents able tofacilitate introduction of the vector in the host cell, include withoutlimitation polycationic polymers (e.g. chitosan, polymethacrylate, PEI,etc), cationic lipids (e.g.DC-Chol/DOPE, transfectam lipofectin nowavailable from Promega) and liposomes.

Recombinant DNA technologies can also be used to improve expression ofthe nucleic acid molecule in the host cell, e.g. by using high-copynumber vectors, substituting or modifying one or more transcriptionalregulatory sequences (e.g. promoter, enhancer and the like), optimizingthe codon usage to the host cell, and suppressing negative sequencesthat may destabilize the transcript.

Preferably, the nucleic acid molecule encoding the immune checkpointmodulator is in a form suitable for its expression in a host cell, whichmeans that the nucleic acid molecule is placed under the control of oneor more regulatory sequences, appropriate to the vector, the host celland/or the level of expression desired as described in connection withthe therapeutic gene.

Promoters suitable for expression in E. Coli host cell include, but arenot limited to, the bacteriophage lambda pL promoter, the lac, TRP andIPTG-inducible pTAC promoters. Promoters suitable for expression inyeast include the TEF (Mumberg et al., 1995, Gene 156: 119-22), PGK(Hitzeman et al., 1983, Science 219: 620-5), MF alpha (Inokuchi et al.,1987, Mol. Cell. Biol. 7: 3185-93), CYC-1 (Guarente et al, 1981, Proc.Natl. Acad. Sci. USA 78: 2199), GAL-1, GAL4, GAL10, PHO5,glyceraldehyde-3-phosphate dehydrogenase (GAP or GAPDH), and alcoholdehydrogenase (ADH) (Denis et al., 1983, J. Biol. Chem. 25: 1165)promoters. Inducible eukaryotic promoters regulated by exogenouslysupplied compounds can also be used, including without limitation, thezinc-inducible metallothionein (MT) promoter (Mc Ivor et al., 1987, Mol.Cell Biol. 7: 838-48), the dexamethasone (Dex)-inducible mouse mammarytumor virus (MMTV) promoter, the ecdysone insect promoter (No et al.,1996, Proc. Natl. Acad. Sci. USA 93: 3346-51), thetetracycline-repressible promoter (Gossen et al., 1992, Proc. Natl.Acad. Sci. USA 89: 5547-51), the tetracycline-inducible promoter (Kim etal., 1995, J. Virol. 69: 2565-73), the RU486-inducible promoter (Wang etal., 1997, Nat. Biotech. 15: 239-43) and the rapamycin-induciblepromoter (Magari et al., 1997, J. Clin. Invest. 100: 2865-72). Finally,the promoters described for expression of the therapeutic gene are alsosuitable especially for expression of the one or more immune checkpointmodulator, especially in mammalian cells.

In accordance with the present invention, the immune checkpointmodulator can be modified. Various modifications can be contemplatedsuch as those modifying the amino acid sequence as well as those aimedat increasing its biological half-life, its affinity or its stability.

For example, a signal peptide may be included for facilitating secretionof the immune checkpoint modulator in the cell culture. The signalpeptide is typically inserted at the N-terminus of the proteinimmediately after the Met initiator. The choice of signal peptides iswide and is accessible to persons skilled in the art.

As an additional example, a tag peptide (typically a short peptidesequence able to be recognized by available antisera or compounds) maybe also be added for facilitating purification of the recombinant immunecheckpoint modulator. A vast variety of tag peptides can be used in thecontext of the invention including, without limitation, PK tag, FLAGoctapeptide, MYC tag, HIS tag (usually a stretch of 4 to 10 histidineresidues) and e-tag (U.S. Pat. No. 6,686,152). The tag peptide(s) may beindependently positioned at the N-terminus of the protein oralternatively at its C-terminus or alternatively internally or at any ofthese positions when several tags are employed. Tag peptides can bedetected by immunodetection assays using anti-tag antibodies.

As another example, the glycosylation of the immune checkpoint modulatorcan be altered so as to increase its affinity for its target. Suchmodifications can be accomplished, for example, by mutating one or moreresidues within the site(s) of glycosylation. Alternatively, the type ofglycosylation can be modified, for example, by expression in a host cellwith altered glycosylation machinery. For illustrative purposes,non-glycosylated protein may be expressed in E. coli whereas modulatorlacking fucose on their carbohydrates may be produced in other cellssuch as those described in US 2004-0110704 (lacking the alpha (1,6)fucosyltransferase activity). Such altered glycosylation patterns havebeen described to increase the ADCC ability of antibodies.

Another modification is pegylation, for example, to increase thebiological half-life of the antibody. Methods for pegylating proteinsare known in the art (see e.g. EP154316; EP401384; WO98/15293,WO01/23001, etc).

Another approach that may be pursued in the context of the presentinvention is coupling of the immune checkpoint modulator to an externalagent such as a radiosensitizer agent, a cytotoxic agent and/or alabelling agent. The coupling can be covalent or not. As used herein,the term “radiosensitizer” refers to a molecule that makes cells moresensitive to radiation therapy. Radiosensitizer includes, but are notlimited to, metronidazole, misonidazole, desmethylmisonidazole,pimonidazole, etanidazole, nimorazole, mitomycin C, RSU 1069, SR 4233,E09, RB 6145, nicotinamide, 5-bromodeoxyuridine (BUdR),5-iododeoxyuhdine (IUdR), bromodeoxycytidine, fluorodeoxyuridine (FUdR),hydroxyurea and cisplatin.

As used herein, the term “cytoxic agent” refers to a compound that isdirectly toxic to cells, preventing their reproduction or growth such astoxins (e. g. an enzymatically active toxin of bacterial, fungal, plantor animal origin, or fragments thereof). As used herein, “a labelingagent” refers to a detectable compound. The labeling agent may bedetectable by itself (e. g., radioactive isotope labels or fluorescentlabels) or, in the case of an enzymatic label, may catalyze chemicalmodification of a substrate compound which is detectable.

The methods for recombinantly producing the immune checkpoint modulatorare conventional in the art. Typically such methods comprise (a)introducing the expression vector described herein into a suitableproducer cell to produce a transfected or infected producer cell, (b)culturing in-vitro said transfected or infected producer cell underconditions suitable for its growth, (c) recovering the immune checkpointmodulator from the cell culture, and (d) optionally, purifying therecovered immune checkpoint modulator. In the context of the invention,producer cells include prokaryotic cells, lower eukaryotic cells such asyeast, and other eukaryotic cells such as insect cells, plant andmammalian (e.g. human or non-human) cells. Preferred E. coli cellsinclude without limitation E. coli BL21 (Amersham Biosciences).Preferred yeast producer cells include without limitation S. cerevisiae,S. pombe, Pichia pastoris. Preferred mammalian producer cells includewithout limitation BHK-21 (baby hamster kidney), CV-1 (African monkeykidney cell line), COS (e.g. COS-7) cells, Chinese hamster ovary (CHO)cells, mouse NIH/3T3 cells, mouse NSO myeloma cells, HeLa cells, Verocells, HEK293 cells and PERC.6 cells as well as the correspondinghybridoma cells.

The producer cells can be cultured in conventional fermentationbioreactors, flasks, and petri plates. Culturing can be carried out at atemperature, pH and oxygen content appropriate for a given host cell. Noattempts to describe in detail the various methods known for theproduction of proteins in prokaryote and eukaryote cells will be madehere. Production of the immune checkpoint modulator can be periplasmic,intracellular or preferably secreted outside the producer cell (e.g. inthe culture medium).

If necessary, especially when the immune checkpoint modulator is notsecreted outside the producer cell or where it is not secretedcompletely, it can be recovered by standard lysis procedures, includingfreeze thaw, sonication, mechanical disruption, use of lysing agents andthe like. If secreted, it can be recovered directly from the culturemedium.

The immune checkpoint modulator can then be purified by well-knownpurification methods including ammonium sulfate precipitation, acidextraction, gel electrophoresis, filtration and chromatographic methods(e.g. reverse phase, size exclusion, ion exchange, affinity,phosphocellulose, hydrophobic-interaction or hydroxylapatitechromatography, etc). The conditions and technology used to purify aparticular protein will depend on factors such as net charge, molecularweight, hydrophobicity, hydrophilicity and will be apparent to thosehaving skill in the art. Moreover, the level of purification will dependon the intended use. It is also understood that depending upon theproducer cell, the immune checkpoint modulator proteins can have variousglycosylation patterns, or may be non-glycosylated (e.g. when producedin bacteria) as described herein.

Desirably, the immune checkpoint modulator in use in the presentinvention is at least partially purified in the sense that it issubstantially free of other antibodies having different antigenicspecificities and/or other cellular material. Further, the immunecheckpoint modulator may be formulated according to the conditionsconventionally used in the art (e.g. WO2009/073569).

Another aspect of this invention pertains to the use of nucleic acidmolecule(s) encoding the immune checkpoint modulator(s) describedherein. For example, the immune checkpoint modulator may be delivered tothe subject in the form of a vector expressing the one or more immunecheckpoint modulator. Any of the vectors described herein can be used inthis context.

Combination Therapy

The oncolytic virus and the one or more immune checkpoint modulator(s)may be administered together in a single composition or concurrently inseparate compositions, optionally comprising a pharmaceuticallyacceptable vehicle in addition to a therapeutically effective amount ofsuch active agent(s). Single composition encompasses the case where theoncolytic virus and said one or more immune checkpoint modulator(s) aremixed together (e.g. a mixture of the oncolytic virus and one or moreantibodies or a mixture of the oncolytic virus and one or more vector(s)for expression of the one or more antibodies). Separate compositions ofthe oncolytic virus and said one or more immune checkpoint modulator(s)may be administered at the same time or sequentially, each once orseveral times (separately or in an interspersed manner) and via the sameor different routes.

A “therapeutically effective amount” corresponds to the amount of eachof the active agents (oncolytic virus and of the one or more immunecheck point modulator(s)) comprised in the combination or composition ofthe invention that is sufficient for producing one or more beneficialresults. Such a therapeutically effective amount may vary as a functionof various parameters, in particular the mode of administration; thedisease state; the age and weight of the subject; the ability of thesubject to respond to the treatment; kind of concurrent treatment; thefrequency of treatment; and/or the need for prevention or therapy. Whenprophylactic use is concerned, the combination is administered at a dosesufficient to prevent or to delay the onset and/or establishment and/orrelapse of a pathologic condition (e.g. a proliferative disease such ascancer), especially in a subject at risk. For “therapeutic” use, thecombination of virus and immune checkpoint modulator(s) is administeredto a subject diagnosed as having a pathological condition (e.g. aproliferative disease such as cancer) with the goal of treating thedisease, eventually in association with one or more conventionaltherapeutic modalities. In particular, a therapeutically effectiveamount could be that amount necessary to cause an observable improvementof the clinical status over the baseline status or over the expectedstatus if not treated, e.g. reduction in the tumor number; reduction inthe tumor size, reduction in the number or extent of metastases,increase in the length of remission, stabilization (i.e. not worsening)of the state of disease, delay or slowing of disease progression orseverity, amelioration or palliation of the disease state, prolongedsurvival, better response to the standard treatment, improvement ofquality of life, reduced mortality, etc. A therapeutically effectiveamount could also be the amount necessary to cause the development of aneffective non-specific (innate) and/or specific anti-tumor response.Typically, development of an immune response in particular T cellresponse can be evaluated in vitro, in suitable animal models or usingbiological samples collected from the subject. For example, techniquesroutinely used in laboratories (e.g. flow cytometry, histology) may beused to perform tumor surveillance. One may also use various availableantibodies so as to identify different immune cell populations involvedin anti-tumor response that are present in the treated subjects, such ascytotoxic T cells, activated cytotoxic T cells, natural killer cells andactivated natural killer cells. An improvement of the clinical statuscan be easily assessed by any relevant clinical measurement typicallyused by physicians or other skilled healthcare staff.

The term “pharmaceutically acceptable vehicle” is intended to includeany and all carriers, solvents, diluents, excipients, adjuvants,dispersion media, coatings, antibacterial and antifungal agents,absorption agents and the like compatible with administration in mammalsand in particular human subjects.

Each of the oncolytic virus and the one or more immune check pointmodulator(s) or the composition thereof can independently be placed in asolvent or diluent appropriate for human or animal use. The solvent ordiluent is preferably isotonic, hypotonic or weakly hypertonic and has arelatively low ionic strength. Representative examples include sterilewater, physiological saline (e.g. sodium chloride), Ringer's solution,glucose, trehalose or saccharose solutions, Hank's solution, and otheraqueous physiologically balanced salt solutions (see for example themost current edition of Remington: The Science and Practice of Pharmacy,A. Gennaro, Lippincott, Williams&Wilkins).

In other embodiments, each of the oncolytic virus and the immune checkpoint modulator composition(s) is suitably buffered for human use.Suitable buffers include without limitation phosphate buffer (e.g. PBS),bicarbonate buffer and/or Tris buffer capable of maintaining aphysiological or slightly basic pH (e.g. from approximately pH 7 toapproximately pH 9).

Each of the oncolytic virus and/or the immune check point modulatorcomposition(s) may also contain other pharmaceutically acceptableexcipients for providing desirable pharmaceutical or pharmacodynamicproperties, including for example osmolarity, viscosity, clarity,colour, sterility, stability, rate of dissolution of the formulation,modifying or maintaining release or absorption into an the human oranimal subject, promoting transport across the blood barrier orpenetration in a particular organ.

Each of the oncolytic virus and of the immune check point modulatorcomposition(s) can also comprise one or more adjuvant(s) capable ofstimulating immunity (especially a T cell-mediated immunity) orfacilitating infection of tumor cells upon administration, e.g. throughtoll-like receptors (TLR) such as TLR-7, TLR-8 and TLR-9, includingwithout limitation alum, mineral oil emulsion such as, Freunds completeand incomplete (IFA), lipopolysaccharide or a derivative thereof (Ribiet al., 1986, Immunology and Immunopharmacology of Bacterial Endotoxins,Plenum Publ. Corp., NY, p 407-419), saponins such as QS21 (Sumino etal., 1998, J. Virol. 72: 4931; WO98/56415), imidazo-quinoline compoundssuch as Imiquimod (Suader, 2000, J. Am Acad Dermatol. 43:S6), S-27609(Smorlesi, 2005, Gene Ther. 12: 1324) and related compounds such asthose described in WO2007/147529, cytosine phosphate guanosineoligodeoxynucleotides such as CpG (Chu et al., 1997, J. Exp. Med. 186:1623; Tritel et al., 2003, J. Immunol. 171: 2358) and cationic peptidessuch as IC-31 (Kritsch et al., 2005, J. Chromatogr Anal. Technol.Biomed. Life Sci. 822: 263-70).

In one embodiment, the oncolytic virus and the one or more immunecheckpoint modulator(s) comprised in the combination of the presentinvention may be formulated with the goal of improving their stabilityin particular under the conditions of manufacture and long-term storage(i.e. for at least 6 months, with a preference for at least two years)at freezing (e.g. −70° C., −20° C.), refrigerated (e.g. 4° C.) orambient temperatures. Various virus formulation are available in the arteither in frozen, liquid form or lyophilized form (e.g. WO98/02522,WO01/66137, WO03/053463, WO2007/056847 and WO2008/114021, etc). Solid(e.g. dry powdered or lyophilized) compositions can be obtained by aprocess involving vacuum drying and freeze-drying. For illustrativepurposes, sterile histidine, acetate citrate or phosphate buffers salinecontaining surfactant such as polysorbate 80 and stabilizers such assucrose or mannitol are adapted to the preservation of recombinantantibodies and buffered formulations including NaCl and/or sugar areparticularly adapted to the preservation of viruses (e.g. Tris 10 mM pH8 with saccharose 5% (W/V), Sodium glutamate 10 mM, and NaCl 50 mM orphosphate-buffered saline with glycerol (10%) and NaCl).

In certain embodiments, the immune checkpoint modulator can beformulated to ensure proper distribution or a delayed release in vivo.For example, it can be formulated in liposomes. Biodegradable,biocompatible polymers can be used, such as ethylene vinyl acetate,polyanhydrides, polyglycolic acid, collagen, polyorthoesters, andpolylactic acid. Many methods for the preparation of such formulationsare described by e.g. J. R. Robinson in “Sustained and ControlledRelease Drug Delivery Systems”, ed., Marcel Dekker, Inc., New York,1978.

The appropriate dosage of oncolytic virus and immune checkpointmodulator(s) can be adapted as a function of various parameters and maybe routinely determined by a practitioner in the light of the relevantcircumstances. Suitable dosage of the immune checkpoint modulator(s)varies from about 0.01 mg/kg to about 50 mg/kg, advantageously fromabout 0.1 mg/kg to about 30 mg/kg, desirably from about 0.5 mg/kg toabout 25 mg/kg, preferably from about 1 mg/kg to about 20 mg/kg, morepreferably from about 2 mg/kg to about 15 mg/kg, with a specificpreference for doses from about 3 mg/kg to about 10 mg/kg (e.g. 3 mg/kg,4 mg/kg, 5 mg/kg, 6 mg/kg, 7 mg/kg, 8 mg/kg, 9 mg/kg or 10 mg/kg) whenused systemically. However, doses reduced by a factor of 10 to 100 maybe considered for local intratumoral injection(s) of the immunecheckpoint modulator(s). Suitable dosage for the oncolytic virus variesfrom approximately 10⁵ to approximately 10¹³ vp (viral particles), iu(infectious unit) or pfu (plaque-forming units) depending on the virusand the quantitative technique used. As a general guidance, vacciniavirus doses from approximately 10⁵ to approximately 10¹³ pfu aresuitable, preferably from approximately 10⁶ pfu to approximately 10¹¹pfu, more preferably from approximately 10⁷ pfu to approximately 5×10⁹pfu; doses of approximately 10⁸ pfu to approximately 10⁹ pfu beingparticularly preferred (e.g. dose of 10⁸, 2×10⁸, 3×10⁸, 4×10⁸, 5×10⁸,6×10⁸, 7×10⁸, 8×10⁸, 9×10⁸ or 10⁹ pfu) especially for human use. On thesame line, doses reduced by a factor of 10 to 100 may be considered forlocal intratumoral injection(s) of the oncolytic virus. Individual dosesof 10⁶ to 5×10¹² vp are particularly appropriate for oncolyticadenovirus, preferably from 10⁷ to 10¹² vp, more preferably from 10⁸ to5×10¹¹ vp. The quantity of virus present in a sample can be determinedby routine titration techniques, e.g. by counting the number of plaquesfollowing infection of permissive cells using permissive cells (e.g.BHK-21 or CEF), immunostaining (e.g. using anti-virus antibodies; Carol)et al., 1997, Virology 238: 198-211), by measuring the A260 absorbance(vp titers), or still by quantitative immunofluorescence (iu titers).

Administration

The oncolytic virus and/or the immune check point modulator may beadministered together or separately to the subject and in a single doseor multiple doses. Administrations may be performed by the same ordifferent routes and may take place at the same site or at alternativesites.

Any of the conventional administration routes are applicable in thecontext of the invention including parenteral, topical or mucosalroutes, for each of the active agents comprised in the combination ofthe invention. Parenteral routes are intended for administration as aninjection or infusion and encompass systemic as well as local routes.Common parenteral injection types are intravenous (into a vein),intra-arterial (into an artery), intradermal (into the dermis),subcutaneous (under the skin), intramuscular (into muscle) andintratumoral (into a tumor or at its close proximity). Infusionstypically are given by intravenous route. Mucosal administrationsinclude without limitation oral/alimentary, intranasal, intratracheal,intrapulmonary, intravaginal or intra-rectal route. Topicaladministration can also be performed using transdermal means (e.g. patchand the like). Administrations may use conventional syringes and needles(e.g. Quadrafuse injection needles) or any compound or device availablein the art capable of facilitating or improving delivery of the activeagent(s) in the subject. Preferred routes of administration for theimmune checkpoint modulator(s) include intravenous (e.g. intravenousinjection or infusion), intratumoral and intraperitoneal. Transdermalpatches may also be envisaged. Preferred routes of administration forthe oncolytic virus include intravenous and intratumoral. Localintratumoral inoculations of the oncolytic virus could be advantageouslycombined with local intratumoral injections of the immune checkpointmodulator(s), concomitantly or with different scheduling to expectoptimal abscopal effects on distant metastases or tumor lesions. Thismay also permit to lower effective amounts of each product and also toreduce unwanted side effects.

In a preferred embodiment, the oncolytic virus and the one or moreimmune checkpoint modulator(s) can be administered sequentially, such asthe oncolytic virus being administered first and the immune checkpointmodulator(s) second, or vise-versa (immune checkpoint modulator(s) beingadministered first and oncolytic virus second). If more than one immunecheckpoint modulator(s) is used (e.g. anti-PD-1 and anti-CTLA-4antibodies), they may be administered simultaneously or sequentially, inwhich case the dosage of each antibody administered falls within theranges indicated herein. Furthermore, if more than one dose of thecombination therapy is administered sequentially, the order of thesequential administration can be reversed or kept in the same order ateach time point of administration. Moreover, sequential administrationsmay be combined with concurrent administrations. It is also possible toproceed via sequential cycles of administrations that are repeated aftera rest period. Intervals between each administration can be from severalhours to one year (e.g. 24 h, 48 h, 72 h, weekly, every two weeks,monthly or yearly). Intervals can also be irregular (e.g. following themeasurement of monoclonal antibodies in the patient blood levels). Thedoses can vary for each administration within the range described above.

In the context of the invention, the oncolytic virus may be administeredonce or several time (e.g. 2, 3, 4, 5, 6, 7 or 8 times etc) at a dosewithin the range of from 10⁷ to 5×10⁹ pfu. The time interval betweeneach virus administration can vary from approximately 1 day toapproximately 8 weeks, advantageously from approximately 2 days toapproximately 6 weeks, preferably from approximately 3 days toapproximately 4 weeks and even more preferably from approximately 1 weekto approximately 3 weeks. In combination, the immune check pointmodulator(s) is/are administered once or several time (e.g. 2, 3, 4, 5,6, 7 or 8 times etc) at a dose within the range of from 2 mg/kg to 15mg/kg. The time interval between each administration of the immune checkpoint modulator(s) can vary from approximately 1 day to approximately 8weeks, advantageously from approximately 2 days to approximately 6weeks, preferably from approximately 3 days to approximately 4 weeks andeven more preferably from approximately 3 days to approximately 3 weeks.For illustrative purpose, a preferred administration schedule foripilimumab is 3 mg/kg as an intravenous infusion every 3 weeks for atotal of four doses. In some embodiments, two or more monoclonalantibodies with different binding specificities are administeredsimultaneously, in which case the dosage of each antibody administeredfalls within the ranges indicated. In a preferred embodiment, theoncolytic virus and the immune checkpoint modulator(s) are administeredsequentially (separately or interspersed), with a specific preferencefor the virus starting first followed by the immune checkpointmodulator(s). The period of time between the first administration of theoncolytic virus and the first administration of the immune check pointmodulator(s) may vary from approximately several hours (at least 6hours) to several week(s). In a preferred embodiment, the method of thepresent invention comprises at least one administration of the oncolyticvirus before starting administration of the immune checkpointmodulator(s), with a specific preference for at least 2 viraladministrations followed by 2 to 5 administrations of the immune checkpoint modulator(s) (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14days separating the second viral administration from the first immunecheckpoint modulator administration). Another preferred therapeuticscheme involves from 2 to 5 (e.g. 3) intravenous or intratumoraladministrations of 10⁸ or 10⁹ pfu of oncolytic vaccinia virus atapproximately 1 or 2 weeks interval followed by or interspersed with 2to 5 (e.g. 3 or 4) intravenous administrations of 3 to 10 mg/kg ofanti-immune checkpoint antibody(ies)(s) every 2 or 3 weeks.

The present invention also relates to a method for treating aproliferative disease such as cancer comprising administering anoncolytic virus and one or more immune checkpoint modulator(s) to asubject in need thereof. In particular, the combination of an oncolyticvirus and one or more immune checkpoint modulator(s) as described hereinis for use for treating a proliferative disease and, especially, fortreating a cancer in a subject having or at risk of having a cancer.

The present invention also relates to a method for inhibiting tumor cellgrowth in vivo comprising administering an oncolytic virus and one ormore immune checkpoint modulator(s) to a subject in need thereof. Inparticular, the combination of an oncolytic virus and one or more immunecheckpoint modulator(s) as described herein is for use for increasinglysis of dividing cells.

The present invention also relates to a method for enhancing an immuneresponse to tumor cells comprising administering an oncolytic virus andone or more immune checkpoint modulator(s) to a subject in need thereof.In particular, the combination of an oncolytic virus and one or moreimmune checkpoint modulator(s) as described herein is for use forincreasing the number and/or functionality of CD8+ T lymphocytes andespecially of tumor-infiltrating CD8+ T lymphocytes.

The present invention also relates to an allogeneic tumor cell lineinfected ex vivo with the oncolytic virus described herein incombination with the one or more immune checkpoint modulator(s)described herein as well as to a method for ex vivo treating aproliferative disease such as cancer comprising administering saidallogenic tumor cell line infected with said oncolytic virus followed byadministering said one or more immune checkpoint modulator(s) to asubject in need thereof, so as to activate the immunity induced by saidinfected allogenic tumor cell line in said subject.

Examples of proliferative diseases that may be treated using thecombination and methods of the invention include bone cancer, livercancer, pancreatic cancer, stomach cancer, colon cancer, cancer of theesophagus, oro-pharyngeal cancer, lung cancer, cancer of the head orneck, skin cancer, melanoma, uterine cancer, cervix cancer, ovariancancer, breast cancer, rectal cancer, cancer of the anal region,prostate cancer, lymphoma, cancer of the endocrine system, cancer of thethyroid gland, sarcoma of soft tissue, chronic or acute leukemias,cancer of the bladder, renal cancer, neoplasm of the central nervoussystem (CNS), glioma, etc. The present invention is also useful fortreatment of metastatic cancers, especially metastatic cancers thatexpress PD-L1 (Iwai et al., 2005, Int. Immunol. 17: 133-44). Preferredcancers that may be treated using the combination therapy according tothe invention include cancers typically responsive to immunotherapy.Non-limiting examples of preferred cancers for treatment includemelanoma (e.g. metastatic malignant melanoma), renal cancer (e.g. clearcell carcinoma), prostate cancer (e.g. hormone refractory prostateadenocarcinoma), breast cancer, colorectal cancer, lung cancer (e.g.non-small cell lung cancer) and liver cancer (e.g. hepatocarcinoma).

According to an advantageous embodiment, especially when the oncolyticvirus is armed with a suicide gene, the combination therapy or methodsaccording to the present invention may comprise an additional step inwhich pharmaceutically acceptable quantities of a prodrug,advantageously an analog of cytosine, in particular 5-FC, areadministered to the subject. By way of illustration, it is possible touse a dose of from 50 to 500 mg/kg/day, with a dose of 200 mg/kg/day orof 100 mg/kg/day being preferred. Within the context of the presentinvention, the prodrug is administered in accordance with standardpractice (e.g. per os, systematically, etc). Preferably, theadministration taking place subsequent to the administration of theoncolytic virus, preferably at least 3 days, more preferably at least 4days and even more preferably at least 7 days after the administrationof the virus. The oral route is preferred. It is possible to administera single dose of prodrug or doses which are repeated for a time which issufficiently long to enable the toxic metabolite to be produced withinthe host organism or cell.

The combination or method according to the invention can be associatedwith one or more substances effective in anticancer therapy. Amongpharmaceutical substances effective in anticancer therapy which may beused in association or in combination with the compositions according tothe invention, there may be mentioned more specifically:

-   -   alkylating agents such as e.g. mitomycin C, cyclophosphamide,        busulfan, ifosfamide, isosfamide, melphalan, hexamethylmelamine,        thiotepa, chlorambucil, or dacarbazine;    -   antimetabolites such as, e.g. gemcitabine, capecitabine,        5-fluorouracil, cytarabine, 2-fluorodeoxy cytidine,        methotrexate, idatrexate, tomudex or trimetrexate;    -   topoisomerase II inhibitors such as, e.g. doxorubicin,        epirubicin, etoposide, teniposide or mitoxantrone;    -   topoisomerase I inhibitors such as, e.g. irinotecan (CPT-11),        7-ethyl-10-hydroxy-camptothecin (SN-38) or topotecan;    -   antimitotic drugs such as, e.g., paclitaxel, docetaxel,        vinblastine, vincristine or vinorelbine;    -   platinum derivatives such as, e.g., cisplatin, oxaliplatin,        spiroplatinum or carboplatinum;    -   inhibitors of tyrosine kinase receptors such as sunitinib        (Pfizer) and sorafenib (Bayer); and    -   anti-neoplastic antibodies.

The combination or method according to the invention may also be used inassociation with one or more other agents including but not limited toimmunomodulatory agents such as, e.g. alpha, beta or gamma interferon,interleukin (in particular IL-2, IL-6, IL-10 or IL-12) or tumor necrosisfactor; agents that affect the regulation of cell surface receptors suchas, e.g. inhibitors of Epidermal Growth Factor Receptor (in particularcetuximab, panitumumab, zalutumumab, nimotuzumab, matuzumab, gefitinib,erlotinib or lapatinib) or inhibitors of Human Epidermal Growth FactorReceptor-2 (in particular trastuzumab); and agents that affectangiogenesis such as, e.g. inhibitor of Vascular Endothelial GrowthFactor (in particular bevacizumab or ranibizumab).

Such substances effective in anticancer therapy may be administered tothe subject sequentially or concomitantly with the combination or methodaccording to the invention.

Alternatively or in combination, the combination or method according tothe invention can also be used in association with radiotherapy.

The present invention also provides kits including the active agent(s)of the combination of the invention in kit form. In one embodiment, akit includes at least an oncolytic virus as discussed herein in onecontainer (e.g., in a sterile glass or plastic vial), and one or moreimmune checkpoint modulator(s) as described herein in another container(e.g., in a sterile glass or plastic vial). A preferred kit comprises anoncolytic vaccinia virus (e.g. a vaccinia virus defective for both TKand RR activities armed with a suicide gene) and an immune checkpointmodulator(s) which specifically binds CTLA-4 (e.g. an anti-CTLA-4antibody, such as ipilimumab). Another preferred kit comprises anoncolytic vaccinia virus (e.g. a vaccinia virus defective for both TKand RR activities armed with a suicide gene) and an immune checkpointmodulator(s) which specifically binds PD-1 (e.g., an anti-PD-1 antibody,such as nivolumab or lanbrolizumab). Another preferred kit comprises anoncolytic vaccinia virus (e.g. a vaccinia virus defective for both TKand RR activities armed with a suicide gene) and an immune checkpointmodulator(s) which specifically binds PD-L1 (e.g., an anti-PD-L1antibody, such as MPDL3280A or BMS936559). Optionally, the kit caninclude a device for performing the administration of the active agents.The kit can also include a package insert including informationconcerning the compositions or individual component and dosage forms inthe kit.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1 A, B and C illustrate the growth of MCA205 tumors implanted inmice at different time points (D2, D13 and D20, respectively) treatedwith 2 intratumoral injections of 10⁷ pfu of WRTG17137 at day 0 and 3and 3 intraperitoneal injections of 250 μg of anti-PD-1 antibody at day6, 9 and 12.

FIG. 2 illustrates the percent of survival in mice implanted with 8×10⁵MCA205 tumor cells and treated with 2 intratumoral injections ofWRTG17137 at day 0 and 3 and 3 intraperitoneal injections of anti-PD-1antibody at day 6, 9 and 12.

FIGS. 3 A, B and C illustrate the growth of MCA205 tumors implanted inmice at different time points (D3, D8 and D13, respectively) treatedwith 2 intratumoral injections of 10⁷ pfu of WRTG17137 at day 0 and 3and 3 intraperitoneal injections of 100 μg of anti-CTLA4 antibody at day6, 9 and 12.

FIG. 4 illustrates the percent of survival in mice implanted with 8×10⁵tumor cells and treated with 2 intratumoral injections of WRTG17137 atday 0 and 3 and 3 intraperitoneal injections of anti-CTLA-4 antibody atday 6, 9 and 12.

FIGS. 5 A and B illustrate the growth of MCA205 tumors implanted in micetreated with 2 intratumoral injections of increasing doses of WRTG17137(10⁵, 10⁶ or 10⁷ pfu) at day 0 and 3 and 3 intraperitoneal injections of250 μg of anti-PD-1 antibody at day 6, 9 and 12. Tumor progression ismeasured at days 12 and 14. Black and round points represent tumorprogression in mice treated with the virus only and grey and squarepoints represent tumor progression in mice treated with the virus andthe anti-PD1 antibody.

FIG. 6 illustrates the growth of MCA205 tumors implanted in mice treatedwith 2 intratumoral injections of 10⁷ pfu of WRTG17137 at day 0 and 3and 3 intraperitoneal injections of 250 μg of anti-PD-1 antibody orisotype control, the first antibody injection being 1, 3, 5 or 7 daysafter the second virus injection. Tumor progression is measured overtime. Black round points represent tumor progression in mice treatedwith the virus and isotype control whereas grey square, triangle,diamond and hexagon shaped points represent tumor progression in micetreated with the virus and the anti-PD1 antibody 1, 3, 5 and 7 daysafter the second virus injection.

FIG. 7 illustrates the growth of MCA205 tumors implanted in mice treatedwith 2 intratumoral injections of 10⁷ pfu of WRTG17137 at day 0 and 3and 3 intraperitoneal injections of 100 μg of anti-CTLA-4 antibody orisotype control, the first antibody injection being 1, 3, 5 or 7 daysafter the second virus injection. Tumor progression is measured overtime. Black round points represent tumor progression in mice treatedwith the virus and isotype control whereas grey square, triangle,diamond and hexagon shaped points represent tumor progression in micetreated with the virus and the anti-CTLA-4 antibody 1, 3, 5 and 7 daysafter the second virus injection.

EXAMPLES

We set out to combine immune checkpoint blocking approaches withoncolytic vaccinia vectors. Virus replication in tumors would lead tocell death, destruction of the tumor and liberation of tumor antigen.Combination of oncolytic viruses with anti-immune checkpoint inhibitorsshould release the brakes from T cell generation and resultingtumor-specific T-cells. Preclinical evidence for synergistic effects ofimmune checkpoint blockers combined with viral vectors was to bedemonstrated in mouse tumor models. This implies the use of i)murine-specific anti-immune check point antibodies and ii) an oncolyticpoxvirus capable of infecting murine cells with a higher efficacy.

The oncolytic virus chosen for these studies (WRTG17137) is a vacciniavirus (VV) Western Reserve (WR) strain defective for thymidine kinase(TK) (locus J2R) and RR⁻ (locus 14L) rendering the virus non-replicativein healthy (non-dividing) cells. In contrast, the VV TK⁻RR⁻ is supposedto selectively and efficiently replicate in tumor cells. It is armedwith the chimeric yeast-derived gene FCU-1, an enzyme turning prodrug5-fluorocytosine (5-FC) in the toxic anabolites 5-fluorouracil (5-FU)and 5-fluorouridine-5′monophosphate (Erbs et al., 2000, Cancer Res.,60(14): 3813-22).

Two immune checkpoint modulators, namely anti-PD-1 and anti-CTLA4monoclonal antibodies, were individually tested in combination withWRTG17137.

Combination of Oncolytic VV with Anti-PD-1 MAb

It was first chosen to target the immune checkpoint blocker murine PD-1(mPD-1) with an appropriate antibody. The rat anti mPD-1 antibodyRMP1-14 (BioXcell) was chosen as anti mPD-1. This antibody was shown toblock the interaction of mPD1 with its ligands (Yamazaki et al., 2005,J. Immunol. 175(3): 1586-92).

The combination of mPD-1 inhibitors (commercial clone RMP1-14) with theoncolytic virus WRTG17137 was tested in vivo in the MCA205 (Shu andRosenberg, 1985, Cancer Res. 45(4): 1657-62) mouse model. Variousschedules of administration were experimented.

In a first setting, C57BL/6 mice were subcutaneously injected with 8×10⁵MCA205 tumor cells. Day 7 after tumor cell injection, 250 μg anti mPD1antibody RMP1-14 or its isotype control 2A3 were injectedintraperitoneal (ip) at days 0, 3 and 6. Virus WRTG17137 (1×10⁷ pfu) wasthen injected intratumorally (it) twice at days 7 and 10. Four groups of13 mice were tested, a control group receiving isotype control (3 ipinjections at days 0, 3 and 6), a group of mice treated with theanti-PD-1 mAb (3 ip injections at days 0, 3 and 6), a group of micetreated with the oncolytic virus (2 it injections at days 7 and 10) andthe fourth group receiving both the anti-PD-1 mAb (3 ip injections atdays 0, 3 and 6) followed one day after the last antibody injection by 2injections of WRTG17137 (2 it injections at days 7 and 10). Tumorprogression and mice survival were followed over 40 days.

As expected, tumor increased in size very rapidly in control groupwhereas the tumor growth was delayed in all the three other groupswithin the same extend although a slight improvement was seen in thegroup receiving both mPD-1 antibody and the oncolytic virus. Results ofsurvival are on the same line with a 50% survival obtained at 16, 23, 24and 26 days, respectively in control group, antibody group,WRTG17137-treated group and antibody+ WRTG17137-treated group.

In the second setting, C57BL/6 mice were subcutaneously injected with8×10⁵ MCA205 tumor cells as before and the animals were divided in fourgroups of 13 mice, respectively a control group receiving isotypecontrol (3 ip injections at days 6, 9 and 12), a group of mice treatedwith the oncolytic virus (2 it injections at days 0 and 3 of 1×10⁷ pfuWRTG17137), a group of mice treated with the anti-PD-1 mAb (3 ipinjections at days 6, 9 and 12 of 250 μg anti mPD1 antibody RMP1-14),and the fourth group receiving both the virus (at days 0 and 3) followedthree days after by the antibody (3 ip injections every three days, i.e.at days 6, 9 and 12). Tumor progression and survival were followed over40 days.

As expected, tumors increased in size very rapidly in control groupwhereas the tumor growth was delayed in all the three other groups.However, as illustrated in FIG. 1, tumor growth is delayed within thesame extend in the groups treated with only one component (mPD-1antibody or oncolytic virus) and the slowdown is more pronounced in thegroup receiving both mPD-1 antibody and the oncolytic virus, especiallyat time point D13 and D20.

As illustrated in FIG. 2, 50% survival is obtained at day 16 in thecontrol group. An increase of survival was observed due to WRTG17137injection (50% survival at day 22) or due to mPD-1 injection (50%survival at day 20). Survival was further increased after administrationof WRTG17137 followed by antibody (50% survival measured at day 28).

Combination of Anti-CTLA4 Inhibitors

Combination of anti-CTLA-4 antibody (commercial clone 9D9) with theoncolytic virus WRTG17137 was tested in vivo in the MCA205 mouse model.Various schedules of administration were experimented.

In a first setting, C57BL/6 mice were subcutaneously injected with 8×10⁵tumor cells (MCA205). Day 7 after tumor cell injection, 100 μg antiCTLA-4 antibody 9D9 (BioXcell) were injected ip at days 0, 3 and 6.Virus WRTG17137 (1×10⁷ pfu) was injected intratumorally twice at days 7and 10. Four groups of 6 mice were tested, respectively a control groupreceiving isotype control MCP-11 (3 ip injections at days 0, 3 and 6), agroup of mice treated with the anti-CTLA-4 mAb (3 ip injections at days0, 3 and 6), a group treated with the oncolytic virus (2 it injectionsat days 7 and 10) and the fourth group receiving both the anti-CTLA-4antibody (3 ip injections at days 0, 3 and 6) followed one day after thelast antibody injection by 2 injections of WRTG17137 (2 it injections atdays 7 and 10). Tumor progression and mice survival were followed over35 days

As expected, tumor increased in size very rapidly in control groupwhereas the tumor growth was delayed in all the three other groupswithin approximately the same extend.

In the second setting, mice were subcutaneously injected with 8×10⁵ MCAtumor cells as before. Four groups of 6 mice were tested, a controlgroup receiving the isotype control MCP-11 (3 ip injections at days 0, 3and 6), a group of mice treated with the oncolytic virus (2 itinjections at days 0 and 3 of 1×10⁷ pfu WRTG17137), a group of micetreated with 100 μg of anti-CTLA-4 mAb 9D9 (3 ip injections at days 6, 9and 12), and the fourth group receiving both the virus (at days 0 and 3)followed three days after by the anti-CTLA-4 antibody (3 ip injectionsevery three days, i.e. at days 6, 9 and 12). Tumor progression andsurvival were followed over 35 days.

As expected, tumors increased in size very rapidly in control groups.Tumor growth was delayed in all the three other groups. However, asillustrated in FIG. 3, the tumor volume is decreased in the groupstreated with only one component (anti-CTLA-4 antibody or oncolyticvirus), the slowdown is much more pronounced in the group receiving bothantiCTLA4 antibody and the oncolytic virus.

As illustrated in FIG. 4, 50% survival is obtained at day 18 in thecontrol group. An increase of survival was observed due to WRTG17137injection (50% survival at day 21) or due to anti CTLA-4 antibodyinjection (50% survival at day 20). Survival was further increased afteradministration of WRTG17137 followed by antibody (50% survival measuredat day 32).

Dose Effects

The same experiment as before was carried out with varying doses ofvirus. Four groups of six mice were treated after tumor implantation(8×10⁵ MCA tumor cells). A control group received formulation buffer inplace of virus and isotype control in place of the antibody. A secondgroup was treated with 10⁵, 10⁶ or 10⁷ pfu of WRTG17137 (2 it injectionsat days 0 and 3) and a third one with the anti-PD-1 mAb (3 ip injectionsat days 6, 9 and 12 of 250 μg anti mPD1 antibody RMP1-14). A fourthgroup received both the virus (10⁵, 10⁶ or 10⁷ pfu at days 0 and 3)followed three days after by the antibody (3 ip injections every threedays, i.e. at days 6, 9 and 12). Tumor progression was followed over 15days.

FIG. 5 illustrates the tumor progression observed in mice treated withthe same dose of virus alone (black and round points) or in combinationwith the anti-PD-1 (grey and square points) 12 and 14 days following thefirst virus injection. Whatever the virus dose injected into the tumor,tumor growth is delayed in mice treated with the combination ofvirus+anti-PD-1 as compared to that measured in mice treated with theoncolytic virus only.

These results illustrate the therapeutic and synergistic anti-tumoractivity of the combination of the invention especially when virus isadministered first before the immune check point modulator.

Variation of the Time Interval Between Virus and AntibodyAdministrations

Anti-PD1 Antibody Combination

Six weeks old female C57BL/6 mice were injected subcutaneously (sc) intothe right flanks with 8×10⁵ MCA205 tumor cells. At day 0 (DO), whentumor volumes reached 40-60 mm², the animals were randomized and dividedin 11 groups of 6 mice. A control group receiving buffer (2 itinjections at days 0 and 3), a group of mice treated with the oncolyticvirus (2 it injections at days 0 and 3 of 1×10⁷ pfu WRTG17137), a groupreceiving both the virus (at days 0 and 3) and the isotype control (3 ipinjections at days 6, 9 and 12), a group of mice treated with both thevirus (at days 0 and 3) and the anti-PD-1 mAb (3 ip injections at days4, 7 and 10 of 250 μg anti mPD1 antibody clone RMP1-14), a group of micetreated with both the virus (at days 0 and 3) and the anti-PD-1 mAb (3ip injections at days 6, 9 and 12 of 250 μg anti mPD1 antibody cloneRMP1-14), a group of mice treated with both the virus (at days 0 and 3)and the anti-PD-1 mAb (3 ip injections at days 8, 11 and 14 of 250 μganti mPD1 antibody clone RMP1-14), a group of mice treated with both thevirus (at days 0 and 3) and the anti-PD-1 mAb (3 ip injections at days10, 13 and 16 of 250 μg anti mPD1 antibody clone RMP1-14), a group ofmice receiving both the virus (at days 0 and 3) and the anti-PD-1 mAb (3ip injections at days 4, 7 and 10 of 100 μg anti mPD1 antibody cloneRMP1-14), a group of mice receiving both the virus (at days 0 and 3) andthe anti-PD-1 mAb (3 ip injections at days 6, 9 and 12 of 100 μg antimPD1 antibody clone RMP1-14), a group of mice receiving both the virus(at days 0 and 3) and the anti-PD-1 mAb (3 ip injections at days 8, 11and 14 of 100 μg anti mPD1 antibody clone RMP1-14), and a group of micereceiving both the virus (at days 0 and 3) and the anti-PD-1 mAb (3 ipinjections at days 10, 13 and 16 of 100 μg anti mPD1 antibody cloneRMP1-14). Tumor progression and survival were followed over time.

As shown in FIG. 6, tumor growth is delayed in mice treated with thecombination of virus and anti-PD-1 (250 μg/mice) as compared to thatmeasured in mice treated with the oncolytic virus and the isotypeantibody. The more time there is between the administrations of thevirus and those of antibody, the more tumor growth is controlled.Statistical differences between the control group and thevirus+anti-PD-1 group (D+7, that is 3 ip injections at days 10, 13 and16 of 250 μg anti mPD1 antibody clone RMP1-14) were seen at 9 and 13days post treatment. The same tendency was observed in the groups ofanimals treated with the combination of WRTG17137 (1×10⁷ pfu/mice) andanti-PD-1 (100 μg/mice) although without any statistical differenceswith respect to the control group (1×10⁷ pfu/mice+100 μg isotype/mice).

Anti-CTLA-4 Antibody Combination

Six weeks old female C57BL/6 mice were injected subcutaneously (sc) intothe right flanks with 8×10⁵ MCA205 tumor cells. At day 0 (DO), whentumor volumes reached 40-60 mm², the animals were randomized and dividedin 11 groups of 6 mice. A control group receiving buffer (2 itinjections at days 0 and 3), a group of mice treated with the oncolyticvirus (2 it injections at days 0 and 3 of 1×10⁷ pfu WRTG17137), a groupreceiving both the virus (at days 0 and 3) and the isotype control (3 ipinjections at days 6, 9 and 12), a group of mice treated with both thevirus (at days 0 and 3) and the anti-CTLA4 mAb (3 ip injections at days4, 7 and 10 of 100 μg anti mCTLA4 antibody clone 9D9), a group of micetreated with both the virus (at days 0 and 3) and the anti-CTLA4 mAb (3ip injections at days 6, 9 and 12 of 100 μg anti mCTLA4 antibody clone9D9), a group of mice treated with both the virus (at days 0 and 3) andthe anti-CTLA4 mAb (3 ip injections at days 8, 11 and 14 of 100 μg antim CTLA4 antibody clone 9D9), a group of mice treated with both the virus(at days 0 and 3) and the anti-CTLA4 mAb (3 ip injections at days 10, 13and 16 of 100 μg anti-mCTLA4 antibody clone 9D9), a group of micereceiving both the virus (at days 0 and 3) and the anti-CTLA4 mAb (3 ipinjections at days 4, 7 and 10 of 50 μg anti-mCTLA4 antibody clone 9D9),a group of mice receiving both the virus (at days 0 and 3) and theanti-CTLA4 mAb (3 ip injections at days 6, 9 and 12 of 50 μg anti-mCTLA4antibody clone 9D9), a group of mice receiving both the virus (at days 0and 3) and the anti-CTLA4 mAb (3 ip injections at days 8, 11 and 14 of50 μg anti-mCTLA4 antibody clone 9D9), and a group of mice receivingboth the virus (at days 0 and 3) and the anti-CTLA4 mAb (3 ip injectionsat days 10, 13 and 16 of 50 μg anti-mCTLA4 antibody clone 9D9). Tumorprogression and survival were followed over time.

As shown in FIG. 7, tumor growth is delayed in mice treated with thecombination of virus and anti-CTLA-4 (100 μg/mice) as compared to thatmeasured in mice treated with the oncolytic virus and the isotypeantibody. Tumor growth was even more delayed with short time period (day1, 3 or 5 days) between the virus administrations and the antibodyadministrations with statistical differences observed for these 3 groupswith respect to the control group at 6, 9 and 13 days post treatment.The same tendency was observed in the D+1, D+3 and D+5 groups of animalstreated with the combination of WRTG17137 (1×10⁷ pfu/mice) andanti-CTLA-4 (50 μg/mice), although without any statistical differenceswith respect to the control group (1×10⁷ pfu/mice+100 μg isotype/mice).

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, numerous equivalents to thespecific method and reagents described herein, including alternatives,variants, additions, deletions, modifications and substitutions. Suchequivalents are considered to be within the scope of this invention andare covered by the following claims.

The invention claimed is:
 1. A method for treating a cancer, comprisingadministering: i) an oncolytic vaccinia virus, wherein said oncolyticvaccinia virus is defective for thymidine kinase (TK) resulting frominactivating mutations in the J2R viral gene and is defective forRibonucleotide reductase (RR) activity resulting from inactivatingmutations in the viral I4L and/or F4L gene(s) and ii) one or more immunecheckpoint modulator(s) consisting of an antibody, wherein the antibodyspecifically binds to PD-1 and is selected from Nivolumab andPembrolizumab, wherein said cancer is selected from the group consistingof: bone cancer, liver cancer, pancreatic cancer, stomach cancer, coloncancer, cancer of the esophagus, oro-pharyngeal cancer, lung cancer,cancer of the head or neck, skin cancer, melanoma, uterine cancer,cervix cancer, ovarian cancer, breast cancer, rectal cancer, cancer ofthe anal region, prostate cancer, lymphoma, cancer of the endocrinesystem, cancer of the thyroid gland, sarcoma of soft tissue, chronic oracute leukemias, cancer of the bladder, renal cancer, neoplasm of thecentral nervous system (CNS), and glioma, wherein said oncolyticvaccinia virus and said one or more immune checkpoint modulator(s) areadministered sequentially and wherein said oncolytic vaccinia virus isadministered first and said immune checkpoint modulator(s) isadministered second.
 2. The method of claim 1, wherein said oncolyticvaccinia virus further expresses at least one therapeutic gene insertedin the viral genome, wherein said therapeutic gene is selected from thegroup consisting of genes encoding suicide gene products and genesencoding immunostimulatory proteins.
 3. The method of claim 2, whereinsaid suicide gene is selected from the group consisting of genesencoding a protein having a cytosine deaminase (CDase) activity, athymidine kinase activity, an uracil phosphoribosyl transferase(UPRTase) activity, a purine nucleoside phosphorylase activity and athymidylate kinase activity.
 4. The method of claim 3, wherein saidsuicide gene product has CDase and UPRTase activities.
 5. The method ofclaim 4, wherein said oncolytic vaccinia virus is defective for both TKand RR activities and comprising inserted into its genome thetherapeutic FCU1 suicide gene.
 6. The method of claim 2, wherein saidimmunostimulatory protein is an interleukin or a colony-stimulatingfactor.
 7. The method of claim 6, wherein said oncolytic vaccinia virusis defective for TK activity and comprises inserted into its genome thetherapeutic human GM-CSF.
 8. The method according to claim 1 comprisingfrom approximately 10⁷ pfu to approximately 5×10⁹ pfu of said oncolyticvaccinia virus.
 9. The method of claim 1, comprising from about 2 mg/kgto about 15 mg/kg of said one or more immune checkpoint modulator(s).10. The method of claim 1, wherein said immune checkpoint modulator(s)is administered by intravenous, intratumoral or intraperitoneal routeand wherein said oncolytic vaccinia virus is administered by intravenousor intratumoral route.
 11. The method of claim 1, which comprises from 2to 5 intravenous or intratumoral administrations of 10⁸ or 10⁹ pfu ofoncolytic vaccinia virus at approximately 1 or 2 weeks interval followedby or interspersed with 2 to 5 intravenous administrations of 3 to 10mg/kg of one or more anti-immune checkpoint antibody(ies)(s) every 2 or3 weeks.
 12. A kit comprising: i) in one container an oncolytic vacciniavirus, wherein said oncolytic vaccinia virus is defective for thymidinekinase (TK) resulting from inactivating mutations in the J2R viral geneand is defective for Ribonucleotide reductase (RR) activity resultingfrom inactivating mutations in the viral 14L and/or F4L gene(s); ii) inanother container one or more immune checkpoint modulator(s) consistingof an antibody, wherein the antibody specifically binds to PD-1 and isselected from Nivolumab and Pembrolizumab; and iii) instructions for useindicating that said oncolytic vaccinia virus and said one or moreimmune checkpoint modulator(s) are to be administered sequentially andthat said oncolytic vaccinia virus is to be administered first and saidimmune checkpoint modulator(s) is to be administered second.
 13. Apharmaceutical composition comprising: i) an oncolytic vaccinia virus,wherein said oncolytic vaccinia virus is defective for thymidine kinase(TK) resulting from inactivating mutations in the J2R viral gene and isdefective for Ribonucleotide reductase (RR) activity resulting frominactivating mutations in the viral 14L and/or F4L gene(s); and ii) oneor more immune checkpoint modulator(s) consisting of an antibody,wherein the antibody specifically binds to PD-1 and is selected fromNivolumab and Pembrolizumab.
 14. A method for treating a cancer,comprising administering: i) an oncolytic vaccinia virus, wherein saidoncolytic vaccinia virus is defective for thymidine kinase (TK)resulting from inactivating mutations in the J2R viral gene and isdefective for Ribonucleotide reductase (RR) activity resulting frominactivating mutations in the viral I4L and/or F4L gene(s), and whereinsaid oncolytic vaccinia virus is approximately 10⁷ pfu to approximately5×10⁹ pfu; and ii) an antibody selected from Nivolumab andPembrolizumab, wherein said antibody is about 1 mg/kg to about 20 mg/kg;wherein said cancer is selected from the group consisting of: bonecancer, liver cancer, pancreatic cancer, stomach cancer, colon cancer,cancer of the esophagus, oro-pharyngeal cancer, lung cancer, cancer ofthe head or neck, skin cancer, melanoma, uterine cancer, cervix cancer,ovarian cancer, breast cancer, rectal cancer, cancer of the anal region,prostate cancer, lymphoma, cancer of the endocrine system, cancer of thethyroid gland, sarcoma of soft tissue, chronic or acute leukemias,cancer of the bladder, renal cancer, neoplasm of the central nervoussystem (CNS), and glioma; wherein said oncolytic vaccinia virus and saidantibody are administered sequentially; and wherein said oncolyticvaccinia virus is administered first and said antibody is administeredsecond.